Virus coat protein/receptor chimeras and methods of use

ABSTRACT

The invention relates to chimeric molecules comprising a virus coat sequence and a receptor sequence that can inter-act with each other to form a complex that is capable of binding a co-receptor. Such chimeric molecules therefore exhibit functional properties characteristic of a receptor-coat protein complex and are useful as agents that inhibit virus infection of cells due to occupancy of a co-receptor present on the cell. In particular aspects, the chimeric polypeptide includes an immunodeficiency virus envelope polypeptide, such as that of HIV, SIV, FIV, FeLV, FPV and herpes virus. Receptor sequences suitable for use in a chimeric polypeptide include, for example, CD4 D1D2 and CD4M9 sequences.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisonal of U.S. patent application Ser. No.09/934,060 filed on Aug. 21, 2001 which claims priority from co-pendingapplication U.S. Ser. No. 09/684,026 filed on Oct. 6, 2000 that claimspriority from U.S. Provisional application No. 60/158,321 filed on Oct.8, 1999.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under R0 1 HL59796awarded by National Institutes of Health. The Government may havecertain rights in the invention.

BACKGROUND THE INVENTION

1. Field of the Invention

This invention relates generally to receptor ligand interactions, andmore specifically to chimeric polypeptides having virus coat polypeptideand cell receptor polypeptide sequences that bind to each other andmimic the structural, functional and immunogenic properties thatnaturally occur when the virus protein and receptor interact in vivo.

2. Description of Related Art

Humoral immunity arising after primary infection with HIV-1 may notprevent progression to AIDS (R. A. Koup et al., Nature, 370:416 (1994);R. A. Koup et al., J. Virol. 68:4650-5 (1994)). However, it is likelythat Humoral immunity can prevent infection if an individual hashigh-titered neutralizing antibodies prior to exposure to the virus.This concept is largely supported by passive immunization studies inwhich chimps were transfused with neutralizing anti-V3 monoclonalantibodies or pooled, high-titered neutralizing antisera around the timeof challenge with cell-free virus (E. A. Emini et al., Nature:355:728-30 (1992); R. Shibata et al., Nat. Med., 5:204-10 (1999)).Protection was obtained in both sets of studies, indicating that humoralimmunity can be protective provided the right antibodies are present insufficient titers at the time of challenge or shortly thereafter.

Additional studies suggest that humoral immunity can be protectiveagainst HIV-1. For example, passive immunization using the SCID-hu mousesystem have shown that human monoclonal antibodies specific for the CD4binding domain of gp120 can prevent infection (M. C. Gauduin et al.,Nat. Med., 3: 1389-93 (1997); P. W. Parren et al., AIDS, 9:F1-6 (1995)).It has been further shown that passive transfer of a bivalent CD4-Ig“immunoadhesin,” a chimera made between CD4 and the human IgG2 heavychain, can protect in the HIV-1 chimp challenge system (J. W. Eichberget al., AIDS Res. Hum. Retroviruses, 8: 1515-19 (1992); R. H. Ward etal., Nature, 352:434-6 (1991)). Additionally, neutralizing antibodiescorrelate strongly with protective immunity against SIV (J. L. Heeney etal., Proc. Natl. Acad. Sci. US.A., 95: 10803-8 (1998)). Still further,passive transfer studies in rhesus macaques showed that high-titeredchimp antibodies specific for the HIV-1 _(DH12) isolate, providedsterilizing immunity in rhesus macaques against SHIV_(DH12) if asufficient concentration of the antibodies was used (R. Shibata et al.,Nat. Med., 5:204-10 (1999)). Also, passive-transfer experiments inrhesus macaques using HIVIg, 2G12, and 2F5 demonstrated 50% betterprotection in recipient groups compared to non-recipient controlsagainst challenge with SHIV-89.6P (Mascola et al., J. Virol., 73:4009-18(1999)). Thus, these studies support the idea that immunizationstrategies which elicit persistent, high-titered (or highly effective)neutralizing antibody responses of broad specificity may be protective.A successful strategy to reach this goal has been elusive. The subunitformulations of recombinant monomeric or oligomeric HIV envelope thathave been tested elicit neutralizing responses against a narrow range ofisolates (J. P. Moore et al., AIDS, 9:S117-136 (1995); Q. J. Sattentau,Curr. Opin. Immunol., 8:540-5 (1996); R. Wyatt et al., Science,280:1884-8 (1998)).

SUMMARY OF THE INVENTION

The present invention relates to chimeric polypeptides containing avirus coat polypeptide sequence and a viral receptor polypeptidesequence in which the coat polypeptide sequence and the receptorpolypeptide sequence are linked by a spacer. The coat polypeptide andthe viral receptor polypeptide sequences of the chimeric polypeptidescan bind to each other. The chimeric polypeptides of the invention areuseful for inducing an immune response and for producing antibodies.Further, the chimeric polypeptides are useful for preventing,inhibiting, or ameliorating a viral infection by passive protectionagainst virus infection or by production of an immune response (i.e.,antibodies or a CTL response) by administration to a subject.

In various embodiments, the virus coat polypeptide sequence of achimeric polypeptide is an envelope polypeptide sequence (e.g.,full-length gp120 or a fragment), a virus that binds a co-receptorpolypeptide, an immunodeficiency virus, including HIV (e.g., HIV-1 orHIV-2), SIV, FIV, FeLV, FPV, and a herpes virus. In various additionalembodiments, the viral receptor polypeptide sequence is a CD4polypeptide sequence, full-length or a fragment thereof, such as the D1,D2 domains and mutations thereof. Introducing envelope genes derivedfrom viruses that use alternative co-receptors could further expand thepotential of these single chain molecules affording protection fromviral infection of different cell types that express the differentco-receptors.

Chimeric polypeptides having heterologous domains also are provided.Such heterologous domains impart a distinct functionality and includetags, adhesins and immunopotentiating agents. For example, heterologousdomains can have an amino acid sequence, such as a c-myc polypeptidesequence or an immunoglobulin polypeptide sequence (e.g., a heavy chainpolypeptide sequence).

In accordance with the present invention, there are providedpolynucleotide sequences having a nucleic acid sequence encodingchimeric polypeptides. The polynucleotides can be included in anexpression vector and are useful for expressing chimeric polypeptides.

In accordance with the present invention, there are provided antibodiesand functional fragments thereof that bind to the chimeric polypeptidesof the present invention. The antibodies are useful in treatment methodsand in diagnostic methods. Such antibodies can neutralize theimmunodeficiency virus in vitro or in vivo, and can also be useful ininhibiting immunodeficiency virus infection, for example, by passiveprotection. Such antibodies can bind to an epitope produced by thebinding of the virus coat polypeptide sequence and viral receptorpolypeptide sequence. For example, such an epitope can be present on anenvelope polypeptide sequence.

The chimeric polypeptides, polynucleotides and antibodies of the presentinvention are useful for treating viral infection, or for inducing animmune response. Thus, in accordance with the present invention, thereare provided chimeric polypeptides, polynucleotides and antibodies in apharmaceutically acceptable carrier.

Methods for producing an antibody include administering a chimericpolypeptide of the present invention in an amount sufficient for thesubject to produce antibodies to the chimeric polypeptide. Such methodsalso can be useful, for example, for inhibiting or ameliorating virusinfection in a subject, or for passive protection, when the antibody isadministered to a recipient subject.

Methods for inhibiting virus infection in a subject includeadministering an effective amount of a chimeric polypeptide of theinvention, or a polynucleotide encoding same to inhibit virus infectionof a cell. The administered chimeric polypeptide can prevent virusinfection by binding to a viral co-receptor on the cells of the subjector produce a protective immune response. The chimeric polypeptide can beadministered in an amount sufficient to ameliorate the virus infectionin the subject.

A method that produces an immune response can produce an antibodyresponse or a CTL response. The antibodies produced can neutralize theimmunodeficiency virus in vitro. The antibodies also may bind to anepitope exposed by the binding of the two polypeptide sequences of thechimeric polypeptide.

Methods for identifying agents that modulate binding or interactionbetween a virus and a virus co-receptor, and a virus and a virusreceptor, also are provided. In one embodiment, a method includescontacting a chimeric polypeptide having a coat protein of a virus thatbinds to a receptor with a co-receptor polypeptide (e.g., a CCR5 orCXCR4 polypeptide sequence) under conditions allowing the chimericpolypeptide and the co-receptor polypeptide to bind, in the presence andabsence of a test agent, and detecting binding in the presence andabsence of the test agent. Decreased binding in the presence of the testagent identifies an agent that inhibits binding between the virus andthe virus co-receptor polypeptide.

In another embodiment, a method includes contacting a chimericpolypeptide under conditions allowing intramolecular binding within thechimeric polypeptide, in the presence and absence of a test agent, anddetecting intramolecular binding or interaction within the chimericpolypeptide. Decreased binding in the presence of the test agentidentifies an agent that inhibits intramolecular binding or interactionbetween the virus and the virus receptor polypeptide in the chimera. Theagent can be added before or after contacting the chimeric polypeptidewith the virus co-receptor polypeptide. The virus co-receptor orreceptor polypeptide can be present on the surface of an intact cell,which can be present in an animal, such as a non-human primate. Themethods can be performed using an immunodeficiency virus, such as HIV,SIV, and the like. Test agents include a library of agents, such aspeptides, organic molecules, antibodies and fragments thereof,antivirals, virus co-receptors, functional fragments, and peptidemimetics thereof.

Methods for identifying a chimeric polypeptide sequence that modulate(inhibits or stimulates) virus infection of a cell also are provided. Inone embodiment, a method includes contacting a cell susceptible to virusinfection with an infectious virus particle in the presence and absenceof the chimeric polypeptide sequence of the present invention anddetermining whether the chimeric polypeptide modulates (inhibits orstimulates) virus infection of the cell (in vitro or in vivo).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a polynucleotide construct that encodes exemplarychimeric polypeptides. Full-length single chain (FLSC) chimericpolypeptide comprises an HIV gp120 (BaL strain), a 20 amino acid spacerpolypeptide, a CD4 polypeptide sequence comprising the D1 and D2 domains(D1 D2), and a myc peptide “tag.” A truncated single chain (TsSC)chimera contains deletions in the C1 (constant region 1), V1 (variableregion 1), V2, and C5. The deletions indicated for TcSC are numberedaccording to the BaL gp120 sequence. A FLSC R/T chimera has a singlemutation in the furin cleavage site, an R is changed to a T, at thec-terminus of gp120. A FLSC R/T CD4M9 chimera has a single mutation inthe furin cleavage site of gp120, a 21 amino acid spacer polypeptide anda CD4M9 peptide sequence.

FIG. 2 is a Western blot analysis of cell culture supernatant containingFLSC and TcSC soluble chimeric polypeptide expressed by 293-SC cells.Immunoblotting was performed with gp120 (lanes 1 to 4) and CD4 (lanes 5to 8) and the arrows indicate, in order of decreasing gel mobility,gp120-CD4 single chain (single chain), cleaved gp120 (gp120 fragment)and cleaved CD4 (CD4 fragment).

FIG. 3 is an analysis of gp120-CD4 expressed by 293-SC cells;uncrosslinked gp120- CD4 is in lane 1 and the crosslinked gp120-CD4 isin lane 2.

FIG. 4 is an immunoblot analysis of FLSC after crosslinking. Therelative percent (%) total protein for each of the different FLSCconcentrations (1-0.03 uM) are shown in the bar graph: (A), 45% 172 kD;(B), 25% 302 kD; and (C), 10% higher order oligomer.

FIG. 5A-5C is a binding analysis of gp120-CD4 chimera. (A), Full lengthsingle chain (FLSC) incubated with anti-gp120 antibodies (17b, 48d, A32and Cl 1) in comparison to crosslinked gp120/rsCD4 and uncomplexedgp120. 17b, 48d and A32 have preferential affinity for complexed gp120(gp120). Bars are shown with standard error. (B), Reciprocalhalf-maximal binding concentration of human anti-gp120 monoclonalantibodies in FLSC and TcSC (ELISA). (C), Reciprocal half-maximalbinding of monoclonal antibodies IgG1b12, F91 and 205-469, which reactwith the gp 120 CD4 binding domain.

FIG. 6 is an analysis of gp120-CD4 chimera (FLSC, TcSC) binding to CCR5(R5) or CXCR4 (X4) co-receptor expressing L1.2 cells. Control cells thatdo not express CCR5 or CXCR4 are denoted L1.2. Bound complexes weredetected by flow cytometry using 5 ug/ml of anti-CD4 Mab45. The valuesshown are of a representative study performed three times.

FIG. 7 is an analysis of gp120-CD4 (FLSC, TcSC) binding to co-receptorin the presence of gp120 binding antibodies (17b, 48d, A32, C11 and2G12), and a gp41 antibody (F240). L1.2 cells expressed co-receptor CCR5(R5), CXCR4 (X4), or no co-receptor (L1.2), as indicated. Antibody-freecontrols are denoted “+.” Background measurements obtained withuntreated cells are denoted “−.” Bound complexes were detected by flowcytometry using 5 ug/ml Mab45. Results are presented as percent bindingrelative to the mean fluorescence intensity obtained in the matchedcontrol assay. Average values derived from three separate studies areshown. Standard errors are shown with bars.

FIG. 8 is an analysis of HIV-1₂₀₄₄ (an X4-specific isolate) andHIV-_(Ba1), (an R5-specific isolate) virus neutralization by FLSC, TcSC,BaLgp120 and BaLgp120-rsCD4 complexes. U373 cells expressed CD4, eitherR5 or X4, and P-galactosidase regulated by the HIV-1LTR promoter. AnID₉₀ for FLSC and TcSC against HIV-1₂₀₄₄ was not achieved with themaximum concentrations tested and is therefore presented as >10 ug/ml.

FIG. 9 is a diagram of chimeric gp120-CD4-IgG1 gene showing the codingdomains. It is essentially the original gp120-CD4 subcloned into aplasmid that has the IgG1 heavy chain hinge CH2 and CH3 regions therebypermitting expression of chimeric gp120-CD4-IgG1 polypeptide.

FIG. 10 is an immunoblot analysis of a gp120-CD4-IgG1 chimericpolypeptide expressed in 293 cells. The chimeric gp120-CD4-IgG1 wasisolated from culture supernatant (lane 1) and is shown in comparison topurified HIV strain BaL gp120 polypeptide (lane 2). Cleaved gp120 isindicated by the arrow and co-migrates with purified gp120.

FIG. 11 is a reciprocal dilution analysis of gp120-CD4-IgG1 chimericpolypeptide binding to co-receptor expressing L1.2 cells. CCR5 and CXCR4expressing L1.2 cells are as indicated.

FIG. 12 is an analysis of a blocking MAb (17b) on FLSC-IgG1 binding toCCR5 expressing cells showing that FLSC-IgG1 interacts with the R5co-receptor via the R5-binding domain on gp120.

FIG. 13 shows the improved stability of gp120-CD4 (FLSC) moleculesfollowing mutation of furin cleavage site (R-T).

FIG. 14 shows an immunoblot comparing FLSC R/T CD4M9 with BaLgp120,FLSC, and FLSC R/T. The FLSC R/T CD4M9 was constructed by switching theCD4 D1D2 sequence in FLSC R/T for a CD4M9 gene sequence.

FIG. 15 is an analysis of FLSC R/T CD4M9 binding to CCR5 (R5). Resultsof the analysis are shown as mean fluorescence intensity. The figureshows that the FLSC R/T CD4M9 binds to R5 expressing cells with anefficiency equivalent to that of FLSC R/T.

FIG. 16 shows the binding of an epitope that becomes increasing exposedwhen gp120 interacts with CD4 and that the 17b epitope that is exposedis FLSC R/T CD4M9 and equivalent to that of FLSC R/T.

FIG. 17 shows neutralization of primary R5 HIV-1 (92BR020) by sera fromFLSC inoculated mice.

FIG. 18 shows covalent crosslinking of BaLgp120/sCD4 complexes occludesepitopes that are exposed on FLSC.

FIG. 19 shows an immunoblot comparing purified R/T FLSC-IgG1 in reducingand non-reducing conditions.

FIG. 20 shows binding of R/T FLSC-IgG1 to both human and rhesus CCR5.

FIG. 21 shows RANTES competitively inhibits R/T FLSC-IgG1 binding toCCR5.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that a chimericpolypeptide comprising an HIV envelope polypeptide and a CD4 receptorcan form an interacting complex capable of binding to a co-receptor. Inthe chimeric polypeptides of the present invention, HIV gp120 binding toCD4 mimics the envelope protein-CD4 transition state that occurs whenHIV binds CD4 present on cells; gp120 displays conserved epitopesexposed upon complex formation that interact directly with co-receptor,CCR5. Formation of the envelope-CD4 transition state and subsequentbinding to cell co-receptor is a critical step in HIV infection ofcells. Therefore, antibodies or other agents that prevent or inhibitgp120-CD4 binding to co-receptor, for example, by binding epitopesexposed upon gp120-CD4 complex formation could inhibit virus interactionwith the co-receptor thereby mediating protection from HIV infection.

Accordingly, chimeric polypeptides or a nucleic acids encoding thechimeric polypeptides of the present invention can be usedtherapeutically for treating, inhibiting, preventing or amelioratingvirus infection, for example, by inducing an immune response to thetransition state complex formed upon binding of a virus coat protein toa receptor polypeptide. Such chimeric polypeptides, also referred toherein as “single chain” molecules, can be used to screen for agentsthat inhibit, prevent or disrupt the binding of the coat polypeptidesequence to the polypeptide receptor sequence within the chimericsequence, or binding of the chimera to a co-receptor polypeptidesequence, thereby identifying potential therapeutics for treating thecorresponding viral infection. For example, an agent that inhibits,prevents or disrupts immunodeficiency virus envelope polypeptide CD4complex binding to CCR5 can be a therapeutic agent for treating asubject having or at risk of having HIV.

Chimeric polypeptides are also useful for producing antibodies specificfor the interacting coat protein-receptor complex. Such specificantibodies can be used for passive protection against virus infection orproliferation, for diagnostic purposes and for identifying andcharacterizing epitopes exposed upon complex formation (e.g., a crypticepitope). Even in the absence of intramolecular binding between viruscoat protein and a receptor, a chimeric polypeptide may be moreeffective at eliciting an immune response than a virus coat polypeptidesequence alone. Accordingly, such non-interacting chimeric polypeptidesalso are valuable and are included herein.

Chimeric polypeptides containing a virus coat polypeptide that binds areceptor and co-receptor have the additional advantage of passivelyprotecting against virus infection by inhibiting virus access to cellco-receptors in vivo. Moreover, such chimeric polypeptides can be usedto screen for therapeutics by identifying agents that inhibit, preventor disrupt the binding of the chimeric polypeptide to co-receptor. Forexample, an agent that inhibits, prevents or disrupts binding of theimmunodeficiency virus envelope polyeptide-CD4 complex to CCR5 can be atherapeutic agent for treating a subject having or at risk of havingHIV. As virus binding to cell receptors is required for virus infectionof any cell, chimeric polypeptides comprising a polypeptide sequence ofany virus coat protein and a corresponding receptor are included in theinvention compositions and methods.

In accordance with the present invention, there are provided chimericpolypeptides comprising a virus coat polypeptide sequence and a viralreceptor polypeptide sequence linked by a spacer. The coat polypeptidesequence and receptor polypeptide sequence of the chimeric polypeptideare linked by a spacer having a sufficient length of amino acids suchthat the two polypeptide sequences of the chimeric polypeptidepreferably bind or interact. In one embodiment, the coat polypeptidesequence is an envelope polypeptide sequence of an immunodeficiencyvirus. In another embodiment, the coat polypeptide sequence is from avirus that binds a co-receptor polypeptide. In various otherembodiments, the coat polypeptide sequence and the receptor polypeptidesequence are active fragments of a corresponding full-length nativesequence.

As used herein, the term “coat” means a polypeptide sequence of virusorigin that can bind to cells. Generally, virus coat proteins arepresent near the exterior surface of the virus particle and allowbinding and subsequent penetration into the cell membrane. However, acoat polypeptide sequence includes any virus protein capable of bindingto or interacting with a receptor polypeptide. Coat polypeptidesequences as defined herein may be non-covalently or covalentlyassociated with other molecular entities, such as carbohydrates, fattyacids, lipids and the like. Coat polypeptide sequences may containmultiple virus polypeptide sequences. For example, a gag polypeptidesequence may also be included with an envelope polypeptide sequence in achimeric polypeptide to maintain the envelope polypeptide sequence in aconformation that binds to a receptor polypeptide sequence.

Virus coat polypeptide sequences useful in the present invention can beof any origin including, for example, bacterial, plant, and animalviruses, so long as a corresponding cell receptor is known or can beidentified. Examples of particular virus included are: Retroviridae (e.ghuman immunodeficiency viruses, such as HIV); Picornaviridae (e.g.,polio viruses, hepatitis A virus; enteroviruses, human coxsackieviruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains thatcause gastroenteritis); Togaviradae (e.g., equine encephalitis viruses,rubella viruses); Flaviridae (e.g., dengue viruses, encephalitisviruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses);Rhabdoriridae (e.g., vesicular stomatitis viruses, rabies viruses);Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaanviruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae(hemorrahagic fever viruses); Reoviridae (e.g., reoviruses, orbivirusesand rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpessimplex virus (HSV) 2 and 2, varicella zoster virus, cytomegalovirus(CMV), herpes viruses); Poxviridae (variola viruses, vaccinia viruses,pox viruses); and Iridoviridae (e.g., African swine fever virus); andunclassified viruses (e.g., the etiological agents of Spongiformencephalopathies, the agent of delta hepatitis (thought to be adefective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 132 internally transmitted; class 2=parenterallytransmitted (i.e., Hepatitis C); Norwalk and related viruses, andastroviruses). (See also, Table 1).

As used herein, the term “receptor” means any polypeptide expressed by acell that a virus can bind. Generally, such receptors are naturallypresent on the surface of a cell, but can be engineered. Receptorpolypeptides may be non-covalently or covalently associated with othermolecular entities, such as carbohydrates, fatty acids, lipids and thelike. A receptor polypeptide may comprise one or multiple contiguouspolypeptide segments that are covalently or non-covalently attached.Such molecular entities or other polypeptide sequences may be importantfor receptor conformation, for example, for binding to a coatpolypeptide sequence. Thus, additional elements including moleculesimportant for receptor conformation may therefore be included in thechimeric polypeptides of the present invention. The receptor polypeptidesequence can be either prokaryotic or eukaryotic in origin.

If eukaryotic, both plant and animal receptors are contemplated.Preferred animal receptors are mammalian, including human and primates,for example, chimps, apes, macaques, gibbons, orangutans and the like,as well as other animal species, including domestic animals andlivestock. An example of a human receptor is CD4. Other examples ofreceptors include glycosaminoglycan and CD2, CR1. Additional receptorsare known and are applicable in the compositions and methods of theinvention (see, for example, Table 1 J see also “Cellular Receptors ForAnimal Viruses” Eckard Wimmer, ed; Cold Spring Harbor Press (1994)).TABLE 1 Receptor (binding subunit) Virus (family) ReferencesImmunglobulin like Molecules VCAM-1 EMC-D (Picornavaridae) Huber(1994)[CAM-1] (first domain) Major Group HRVs, CAV Colonno et at. (1986) 13,18 and 21 Greve et al. (1989) (picornaviridae) Staunton et al (1989)Tomassini et at. (1989) PVR (first domain) Polioviruses Koike et at.(1990) (Picornaviridae) (1989) Mendelsotm et at. (1989) CD4 (firstdomain) HIV-1, 2; SIV Daigleish et at. (Lentiviridae) (1984); Humanherpesvirus 7 Klatzmaim et at. (1984) Lusso et at. (1994) CEA, severalmember Mouse hepatitis virus Williams et al. (1978) (first domain)(Coronaviridae) MHC 1 Semliki Forest virus Hetenius et at. (1978)(Togaviridae) Otdstone et at. (1980) Factate dehydrogenuse virus Inadaand Mims Mouse cytomegatovirus (1984) (Herpesviridae) Wykes et at.(1993) SV-40 Breau et al. (1992) MHCII Visna virus (Lentiviridae)Dalziel et at. (1991) Integrins VLA-2 (α-chain) ECHO virus 1, 8Bergelson et at. (Picornaviridae) (1992, 1993) (RGD-binding protein)FMDV Fox et at. (1989) (Picornaviridae) Mason et at. (1994) αvβ3(vibronectin) CAV 9, ECHO virus 1.8 Roivainen et at. (Picornaviridae)(1994) Transport proteins Phosphate transporter Gibbon ape leukemiavirus Johann et at. (1992) Analogen (Retroviridae) Miller et al. (1994)Amphotropic murine (Retroviridae) Cationic amino acid Ecotropic murineleukemia Albritton et al. (1989) transporter virus (Retroviridae)Signaling Receptors LDL Receptor protein family Minor group HRVs Hoferet al. (1994) (Picornaviridae) Bates et at. (1993) Subgroup A avianleucosis Connolly et at. (1994) Sarcoma virus (family?) Acetyicholinereceptor (α- 1) Rabies virus (Rhabdoviridae) Leniz (1990) EGF receptorVaccinia virus (Poxviridae) Marsh and Eppstein (1987) Leukocytedifferentiation Feline immunodeficiency Willett et at. (1994) untigen[CD9] Virus (Lentiviridae) Others Aminopeptidase N Human corona virus229E Yeager et at. (1992) (Coronaviridae) Delmas et at. (1992) TGEV(Coronaviridae) Complement receptor CR2 EBV (Herpesviridae) McClure(1992) High affinity laminin receptor Sindbis virus (Togaviridae) Wanget at. (1992) Decay-accelerating factor ECHO viruses 7 Bergelson et at.[CD55] (6, 11, 12, 20, 21) (1994) Membrane cofactor protein Measlesvirus (Morbilliviridae) Dorig et at. (1993) Moesin Measles virus(Morbilliviridae) Dunster et at. (1994) Glycophorin A EMCV(Picornaviridae) Allaway and Barness Reovirus (Reoviridae) (1986) Pauland Lee (1987) Galactosylceramide HIV-1 (Lentiviridae) Bhat et al.(1991) Erythrocyte P antigen Parvovirus B19 Brown et al. (1993)(Parvoviridae) BLV Rcp. 1 Bovine leukemia virus Ban et al. (1993)(Retroviridae) Sialoglycoprotein GP-2 Sendai virus Suzuki et al. (1985)(Paramyxoviridae) Sialic acid Influenza virus Herrler et al. (1985)(Orthomysoviridae) Femandes et al. Reoviridae (Reoviridae) (1994) GroupA porcine rotavirus Roisma et al. (1994) (Rotaviridae) Vlasak et al.(1988) Human coronavirus OC43, bovine coronavirus (Coronaviridae)Heparan sulfate Human cytomegalovirus Compton et al. (1993)(Herpesviridae) WuDunn and Spear HSV (1989)

As used herein, the term “co-receptor” means any receptor that is boundafter or in conjunction with virus binding to receptor. Thus,co-receptors include any polypeptide or molecular entity present on acell that facilitates virus entry, directly or indirectly, by binding tovirus polypeptide-receptor complex. In addition to co-receptors thatfacilitate virus-entry into cells, also included are co-receptors thatmediate cell attachment or tropism without directly or indirectlyfacilitating virus entry. Particular examples of co-receptors are the7-transmembrane domain (7-TM) containing chemokine receptors, such asCCR5 and CXCR4, which can bind immunodeficiency virus. Additionalco-receptors include CCR-2b, CCR3, CCR8, V28/CXCR1, US28, STRL33/BOB/TYMSTR, GPR15/Bonzo and GPR1.

As used herein, the terms “polypeptide,” “protein” and “peptide” areused interchangeably to denote a sequence polymer of at least two aminoacids covalently linked by an amide bond, regardless of length orpost-translational modification (e.g., glycosylation, phosphorylation,lipidation, myristilation, ubiquitination, etc.). D- and L-amino acids,and mixtures of D- and L-amino acids are also included.

Chimeric polypeptide refers to an amino acid sequence having two or moreparts which generally are not found together in an amino acid sequencein nature.

As disclosed herein, a chimeric polypeptide having a CD4 polypeptidesequence and an HIV envelope gp120 polypeptide sequence that binds CD4can bind to each other in the chimera when separated by an amino acidspacer sequence. The gp120-CD4 chimera is capable of binding aco-receptor, such as CCR5. Thus, in another embodiment, the chimericpolypeptide has a coat polypeptide sequence of a virus that binds aco-receptor.

CD4 appears to be the target for entry of a variety of virusesassociated with immunodeficiency. For example, cells of the immunesystem, such as lymphocytes and macrophages express CD4, and aresusceptible to infection by HIV, SIV, herpes virus 7 and many otherviruses. As used herein, the term “immunodeficiency,” when used inreference to a virus, means that the virus is capable of infecting cellsof immune origin or cells that participate in immune responsiveness, andgenerally such infection can compromise an infected host's immunefunction. Thus, the invention is applicable to any virus coatpolypeptide of any virus or virus strain that can bind CD4.

In accordance with the present invention, there are provided chimericpolypeptides having an immunodeficiency virus envelope polypeptidesequence. In various aspects, the envelope polypeptide sequence is apolypeptide sequence of HIV, HTLV, SIV, FeLV, FPV and Herpes virus. Inother aspects, the virus is a macrophage tropic or a lymphocyte tropicHIV. In another aspect, the HIV is HIV-1 or HIV-2. In various otheraspects, the envelope polypeptide sequence is gp120, gp160 or gp41.

Receptor and virus coat polypeptide sequences of the present chimericpolypeptide require a spacer region between them, for example, forforming an interacting complex between the two polypeptides. Althoughnot wishing to be bound by theory, it is believed that the spacer allowsthe movement or flexibility between receptor and virus coat polypeptidesequences to form an interacting complex.

As used herein, the term “spacer” refers to a physical or chemicalmoiety, or covalent or non-covalent bond of any size or nature thatconnects the virus coat polypeptide sequence to the receptor polypeptidesequence while affording the needed flexibility or movement for formingan interacting complex. In the present invention, the spacer preferablylinks the two polypeptide sequences in an “end to end” orientation. “Endto end” means that the amino or carboxyl terminal amino acid of the coatpolypeptide is connected to the amino or carboxyl terminal amino acid ofthe receptor polypeptide sequence. Thus, a spacer can connect thecarboxyl terminal amino acid of the coat polypeptide sequence to theamino terminal amino acid of the receptor polypeptide sequence, asexemplified herein for HIV gp120 and CD4, for example. Alternatively,the spacer can connect the amino terminal amino acid of the coatpolypeptide to the carboxyl terminal amino acid of the receptorpolypeptide or the carboxyl terminal amino acids of the polypeptidesequences or the two amino terminal amino acids of the polypeptidesequences.

Particular examples of spacers include one or more amino acids or apeptidomimetic. An amino acid spacer can essentially be any length, forexample, as few as 5 or as many as 200 or more amino acids. Thus, anamino acid spacer can have from about 10 to about 100 amino acids, orhave from about 15 to about 50 amino acids. Preferably, the spacer hasfrom about 20 to about 40 amino acids. Other examples of spacers includea disulfide linkage between the termini of the polypeptide sequences. Acarbohydrate spacer also is contemplated. Those skilled in the art willknow or can readily ascertain other moieties that can function to allowformation of an interacting complex between the virus coat polypeptidesequence and receptor polypeptide sequence.

Receptor and coat polypeptide sequences can be of any amino acid length.Preferably, they have a length that allows the polypeptide sequences tobind to each other when in a chimeric polypeptide. Thus, receptor andcoat polypeptide sequences include native full-length receptor andfull-length coat polypeptide sequences as well as parts of thepolypeptide sequences. For example, amino acid truncations, internaldeletions or subunits of receptor, and coat polypeptide sequences areincluded. Preferably, such modified forms are capable of interactingwith each other. For example, it is preferable that a truncated ordeleted coat polypeptide sequence is capable of interacting with areceptor polypeptide sequence. An example of a truncated receptorpolypeptide sequence is the CD4 D1 and D2 domains, which are capable ofinteracting with HIV envelope polypeptide sequence (FIG. 9). An exampleof a truncated coat polypeptide sequence is a truncated HIV gp120lacking the amino terminal 60 amino acids and carboxy terminal 20 aminoacids (e.g., in TcSC).

Thus, in accordance with the present invention, chimeric polypeptides,including truncated or internally deleted sequences, are provided. Inone embodiment, the virus coat polypeptide sequence or the receptorpolypeptide sequence has one or more amino acids removed in comparisonto their corresponding full-length polypeptide sequence. In one aspect,the truncated virus coat polypeptide sequence is an HIV envelopepolypeptide sequence and, in another aspect, the truncated receptorpolypeptide sequence is a CD4 sequence. As exemplified herein, thetruncated HIV envelope polypeptide sequence is a gp120 lacking the aminoterminal 60 amino acids or the carboxy terminal 20 amino acids, and atruncated CD4 polypeptide comprising the D1 and D2 domains. In variousother aspects, the chimeric polypeptide comprises an internally deletedvirus coat polypeptide sequence or an internally deleted CD4 polypeptidesequence.

In addition to the truncated, internally deleted and subunit polypeptidesequences, additional polypeptide sequence modifications are included.Such modifications include minor substitutions, variations, orderivitizations of the amino acid sequence of one or both of thepolypeptide sequences that comprise the chimeric polypeptide, so long asthe modified chimeric polypeptide has substantially the same activity orfunction as the unmodified chimeric polypeptide. For example, a viruscoat or receptor polypeptide sequence may have carbohydrates, fattyacids (palmitate, myristate), lipids, be phosphorylated or have otherpost-translational modifications typically associated with polypeptidesequences.

Another example of a modification is the addition of a heterologousdomain that imparts a distinct functionality upon either of the twopolypeptides or the chimeric polypeptide. A heterologous domain can beany small organic or inorganic molecule or macromolecule, so long as itimparts an additional function. Heterologous domains may or may notaffect interaction or affinity between virus coat polypeptide andreceptor polypeptide. Particular examples of heterologous domains thatimpart a distinct function include an amino acid sequence that impartstargeting (e.g., receptor ligand, antibody, etc.), immunopotentiatingfunction (e.g., immunoglobulin, an adjuvant), enable purification,isolation or detection (e.g., myc, T7 tag, polyhistidine, avidin,biotin, lectins, etc.).

Particular heterologous domains may include a c-myc polypeptide sequenceand/or an IgG1 heavy chain polypeptide sequence. A heterologous domaincan have multiple functions. For example, IgG1 can function as animmunopotentiator in vivo, as well as function as an adhesive moleculethat can be purified, isolated, or detected (e.g., by reaction with asecondary antibody having an enzymatic activity, such as horseradishperoxidase or alkaline phosphatase). The skilled artisan will know ofother heterologous domains and can select them as appropriate dependingon the application and the function desired.

Thus, in accordance with the present invention, there are providedchimeric polypeptides having one or more heterologous domains. In oneembodiment, the heterologous domain is a c-myc polypeptide sequenceglu-gln-lys-leu-ile-ser-glu-glu-asp-leu; (SEQ ID NO: 14). In anotherembodiment, the heterologous domain is an immunoglobulin polypeptidesequence comprising a heavy chain (SEQ ID NO: 32).

Receptor and coat polypeptide sequences can be of any amino acid length.Preferably, they have a length that allows the polypeptide sequences tobind to each other when in a chimeric polypeptide. Thus, receptor andcoat polypeptide sequences include native full-length receptor andfull-length coat polypeptide sequences as well as parts of thepolypeptide sequences.

In one aspect, the present invention comprises a full-length singlechain (FLSC) chimeric polypeptide comprising a HIV gp120 (BaL strain),an amino acid spacer polypeptide, a CD4 polypeptide sequence comprisingthe D1D2 domain and a myc peptide “tag” (SEQ ID NO.: 2) or at least 95%sequence identity to SEQ ID NO: 2 that encodes the chimeric polypeptide.

In another aspect, the prevention invention comprises a FLSC polypeptidehaving single mutation in a furin cleavage site of the FLSC polypeptide,wherein an R is changed to a T, at the c-terminus of gp120 (FLSC-R/T) orat least 95% sequence identity to SEQ ID NO: 2 that encodes the chimericpolypeptide. Specifically, FLSC R/T contains an arginine to a threoninemutation at amino acid 506 (SEQ ID NO.: 4).

As exemplified herein, polypeptide sequence include substitutions,variations, or derivitizations of the amino acid sequence of one or bothof the polypeptide sequences that comprise the chimeric polypeptide, solong as the modified chimeric polypeptide has substantially the sameactivity or function as the unmodified chimeric polypeptide. Forexample, a virus coat or receptor polypeptide sequence may havecarbohydrates, fatty acids (palmitate, myristate), lipids, bephosphorylated or have other post-translational modifications typicallyassociated with polypeptide sequences.

In yet another aspect, the virus coat polypeptide sequence or thereceptor polypeptide sequence has one or more amino acid substitutionsin comparison to their corresponding unmodified polypeptide sequences.For example, a nucleotide sequence (SEQ ID NO: 5) is provided thatencodes for a polypeptide that includes a CD4 mimicking receptor thatshows substantially the same activity or improved immune response.Specifically, the gene sequence encoding the amino acid sequence ofKKVVLGKKGDTVELTCTASQKKSIQFHW in CD4 DID2 domain of the chimericpolypeptide FLSC-R/T (SEQ ID NO: 4) is substituted with a nucleotidesequence (SEQ ID NO: 19) that encodes an amino acid sequence ofCNLARCQLRCKSLGLLGKCAGSFCACGP (amino acids 528-556 (SEQ ID NO: 20)) whichis referred to hereinafter as FLSC-R/T CD4M9. (SEQ ID NO.: 6).

As used herein, the term “substantially the same activity or function,”when used in reference to a chimeric polypeptide so modified, means thatthe polypeptide retains most, all or more of the activity associatedwith the unmodified polypeptide, as described herein or known in theart. Similarly, modifications that do not affect the ability of chimericpolypeptide to interact with co-receptor are included herein. Likewise,chimeric polypeptide modifications that do not affect the ability toinduce a more potent immune response than administration of the viruscoat protein alone are included.

Modified chimeric polypeptides that are “active” or “functional”included herein can be identified through a routine functional assay.For example, by using antibody binding assays, co-receptor bindingassays, or determining induction of epitopes exposed in a transitionstate complex normally hidden when the two polypeptide sequences do notbind, one can readily determine whether the modified chimericpolypeptide has activity.

Chimeric polypeptides that induce a more potent immune response can beidentified by measuring antibody titers following administration of thechimera to a subject, for example. Modifications that destroy theinteraction between the virus coat polypeptide sequence and the receptorpolypeptide sequence, or the ability of a chimeric polypeptide having avirus coat polypeptide sequence and receptor sequence which do notinteract to induce a more potent immune response, do not havesubstantially the same activity or function as the corresponding,unmodified chimeric polypeptide and, as such, are not included.

As used herein, the terms “homology” or “homologous,” used in referenceto polypeptides, refers to amino acid sequence similarity between twopolypeptides. When an amino acid position in both of the polypeptides isoccupied by identical amino acids, they are homologous at that position.Thus, by “substantially homologous” means an amino acid sequence that islargely, but not entirely, homologous, and which retains most or all ofthe activity as the sequence to which it is homologous.

As the modified chimeric polypeptides will retain activity or functionassociated with unmodified chimeric polypeptide, modified chimericpolypeptides will generally have an amino acid sequence “substantiallyidentical” or “substantially homologous” with the amino acid sequence ofthe unmodified polypeptide. As used herein, the term “substantiallyidentical” or “substantially homologous,” when used in reference to apolypeptide sequence, means that a sequence of the polypeptide is atleast 50% identical to a reference sequence. Modified polypeptides andsubstantially identical polypeptides will typically have at least 70%,alternatively 85%, more likely 90%, and most likely 95% homology to areference polypeptide. For polypeptides, the length of comparison toobtain the above-described percent homologies between sequences willgenerally be at least 25 amino acids, alternatively at least 50 aminoacids, more likely at least 100 amino acids, and most likely 200 aminoacids or more.

As set forth herein, substantially identical or homologous polypeptidesinclude additions, truncations, internal deletions or insertions,conservative and non-conservative substitutions, or other modificationslocated at positions of the amino acid sequence which do not destroy thefunction of the chimeric polypeptide (as determined by functionalassays, e.g., as described herein). A particular example of asubstitution is where one or more amino acids is replaced by another,chemically or biologically similar residue. As used herein, the term“conservative substitution” refers to a substitution of one residue witha chemically or biologically similar residue. Examples of conservativesubstitutions include the replacement of a hydrophobic residue, such asisoleucine, valine, leucine, or methionine for another, the replacementof a polar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, andthe like. Those of skill in the art will recognize the numerous aminoacids that can be modified or substituted with other chemically similarresidues without substantially altering activity.

Substantially identical or homologous polypeptides also include thosehaving modifications that improve or confer an additional function oractivity. For example, FLSC R/T has a mutated furin site which increasesstability of the modified FLSC (see, e.g., FIG. 13).

Modified polypeptides further include “chemical derivatives,” in whichone or more of the amino acids therein have a side chain chemicallyaltered or derivatized. Such derivatized polypeptides include, forexample, amino acids in which free amino groups form aminehydrochlorides, p-toluene sulfonyl groups, carobenzoxy groups; the freecarboxy groups form salts, methyl and ethyl esters; free hydroxyl groupsthat form O-acyl or O-alkyl derivatives, as well as naturally occurringamino acid derivatives, for example, 4-hydroxyproline, for proline,5-hydroxylysine for lysine, homoserine for serine, ornithine for lysine,and so forth. Also included are D-amino acids and amino acid derivativesthat can alter covalent bonding, for example, the disulfide linkage thatforms between two cysteine residues that produces a cyclizedpolypeptide.

As used herein, the terms “isolated” or “substantially pure,” when usedas a modifier of invention chimeric polypeptides, sequence fragmentsthereof, and polynucleotides, means that they are produced by humanintervention and are separated from their native in vivo-cellularenvironment. Generally, polypeptides and polynucleotides so separatedare substantially free of other proteins, nucleic acids, lipids,carbohydrates or other materials with which they are naturallyassociated.

Typically, a polypeptide is substantially pure when it is at least 60%,by weight, free from the proteins and other molecules with which it isnaturally associated. The preparation is likely at least 75%, morelikely at least 90%, and most likely at least 95%, by weight pure.Substantially pure chimeric polypeptide can be obtained, for example, byexpressing a polynucleotide encoding the polypeptide in cells andisolating the polypeptide produced. For example, as set forth in theexamples, expression of a recombinant polynucleotide encoding agp120-CD4 polypeptide in mammalian cells allows isolating the chimericalpolypeptide from the culture media using an immunoaffinity column.Alternatively, the chimeric polypeptide can be chemically synthesized.Purity can be measured by any appropriate method, e.g., polyacrylamidegel electrophoresis, and subsequent staining of the gel (e.g., silverstain) or by HPLC analysis.

The chimeric polypeptides of the present invention and modificationsthereof can be prepared by a variety of methods known in the art. Thepolypeptide modifications can be introduced by site-directed (e.g., PCRbased) or random mutagenesis (e.g., EMS) by exonuclease deletion, bychemical modification, or by fusion of polynucleotide sequences encodingheterologous domain, for example. Chimeric polypeptides can be obtainedby expression of a polynucleotide encoding the polypeptide in a hostcell, such as a bacteria, yeast or mammalian cell, and purifying theexpressed chimeric polypeptide by purification using typical biochemicalmethods (e.g., immunoaffinity purification, gel purification, expressionscreening etc). Other well-known methods are described in Deutscher etal., (Guide to Protein Purification: Methods in Enzymology, Vol. 182,Academic Press (1990), which is incorporated herein by reference).

The present invention further provides polynucleotide sequences encodingchimeric polypeptides, fragments thereof, and complementary sequences.In one embodiment, nucleic acids encode the chimeric gp120-CD4polypeptide exemplified herein. For example, SEQ ID NO.: 1 defines thesequence encoding FLSC described hereinabove comprising a nucleotidesequence encoding gp120 (SEQ ID 23) and CD4 D1D2 (SEQ ID NO: 25). SEQ.ID NO: 3 defines a sequence encoding FLSC R/T wherein an arginine aminoacid is substituted for a threonine at the c-terminal of the gp120, asuspect furin cleavage site in gp120, thereby improving the stability ofthe FLSC-R/T over FLSC. The nucleotide sequence of FLSC-RT comprises amodified gp120 encoded by SEQ ID NO: 29 and CD4D1D2 (SEQ ID NO: 25).Still further, the present invention provides for polynucleotidesequence SEQ ID NO.: 5 that encodes for a chimeric polypeptide FLSC R/TCD4M9 comprising a substituted furin cleavage site and further providesfor replacement of gene sequence encoding the CD4 D1D2 region with asequence that encodes for an amino acid sequence that mimics a CD4receptor, thereby providing for an improved immune response andadditional stability relative to FLSC or FLSC-R/T. The FLSC R/T CD4M9 isencoded by nucleotide sequences comprising SEQ ID NO: 29 that encodesfor a modified gp120 and SEQ ID NO: 19 encoding for CD4M9. The FLSC R/TCD4M9 chimeric polypeptide may additionally comprise SEQ ID NOs: 23 and19.

In yet another embodiment, TsSC (SEQ ID NO: 12) encode a gp120-CD4polypeptide (SEQ ID NO: 13) in which the gp120 has amino acid sequencestruncated from the amino and carboxy terminus. The nucleotide sequenceof TsSC comprises a sequence (SEQ ID NO: 27) that encodes for atruncated gp120 and CD4D1D2 (SEQ ID NO: 25). In another embodiment, achimeric polypeptide gp120-CD4-IgG1 is encoded by nucleotide SEQ ID NO:1 with an additional tag (SEQ ID NO: 31).

As used herein, the terms “nucleic acid,” “polynucleotide,”“oligonucleotide,” and “primer” are used interchangeably to refer todeoxyribonucleic acid (DNA) or ribonucleic (RNA), either double- orsingle-stranded, linear or circular. RNA can be unspliced or splicedmRNA, rRNA, tRNA, or antisense RNAi. DNA can be complementary DNA(cDNA), genomic DNA, or an antisense. Specifically included arenucleotide analogues and derivatives, such as those that are resistantto nuclease degradation, which can function to encode an inventionchimeric polypeptide. Nuclease resistant oligonucleotides andpolynucleotides are particularly useful for the present nucleic acidvaccines described herein.

An “isolated” or “substantially pure” polynucleotide means that thenucleic acid is not immediately contiguous with the coding sequenceswith either the 5′ end or the 3′ end with which it is immediatelycontiguous in the naturally occurring genome of the organism from whichit is derived. The term therefore includes, for example, a recombinantDNA (e.g., a cDNA or a genomic DNA fragment produced by PCR orrestriction endonuclease treatment produced during cloning), as well asa recombinant DNA incorporated into a vector, an autonomouslyreplicating plasmid or virus, or a genomic DNA of a prokaryote oreukaryote. It also includes a recombinant DNA part of a chimera orfusion, for example. The term therefore does not include nucleic acidspresent but uncharacterized among millions of sequences in a genomic orcDNA library, or in a restriction digest of a library fractionated on agel.

The polynucleotides of the invention also include nucleic acids that aredegenerate as a result of the genetic code. There are 20 natural aminoacids, most of which are specified by more than one codon. Alldegenerate polynucleotide sequences are included that encode inventionchimeric polypeptides.

The polynucleotides sequences of the present invention can be obtainedusing standard techniques known in the art (e.g., molecular cloning,chemical synthesis) and the purity can be determined by polyacrylamideor agarose gel electrophoresis, sequencing analysis, and the like.Polynucleotides also can be isolated using hybridization orcomputer-based techniques that are well known in the art. Suchtechniques include, but are not limited to: (1) hybridization of genomicDNA or cDNA libraries with probes to detect homologous nucleotidesequences; (2) antibody screening of polypeptides expressed by DNAsequences (e.g., using an expression library); (3) polymerase chainreaction (PCR) of genomic DNA or cDNA using primers capable of annealingto a nucleic acid sequence of interest; (4) computer searches ofsequence databases for related sequences; and (5) differential screeningof a subtracted nucleic acid library. Thus, to obtain other receptorencoding polynucleotides, such as those encoding CD4, for example,libraries can be screened for the presence of homologous sequences.

The invention also includes substantially homologous polynucleotides. Asused herein, the term “homologous,” when used in reference to nucleicacid molecule, refers to similarity between two nucleotide sequences.When a nucleotide position in both of the molecules is occupied byidentical nucleotides, then they are homologous at that position.“Substantially homologous” nucleic acid sequences are at least 50%homologous, more likely at least 75% homologous, and most likely 90% ormore homologous. As with substantially homologous invention chimericpolypeptides, polynucleotides substantially homologous to inventionpolynucleotides encoding chimeric polypeptides encode polypeptides thatretain most or all of the activity or function associated with thesequence to which it is homologous. For polynucleotides, the length ofcomparison between sequences will generally be at least 30 nucleotides,alternatively at least 50 nucleotides, more likely at least 75nucleotides, and most likely 110 nucleotides or more. Algorithms foridentifying homologous sequences that account for polynucleotidesequence gaps and mismatched oligonucleotides are known in the art, suchas BLAST (see, e.g., Altschul et al., J. Mol. Biol. 15:403-10 (1990)).

In addition, polynucleotides are useful as hybridization probes in orderto identify the presence or amount of a polynucleotide encoding achimeric polypeptide, for example, mRNA (Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.(1989)). Typically such probes are designed to be specific for thedesired sequence in order to decrease the probability of hybridizing tounrelated sequences. Such probes can be modified so as to be detectableusing radionuclides, luminescent moieties, and so forth. Hybridizationconditions also can be modified in order to achieve the desiredspecificity. For example, a moderately stringent hybridization conditionmay include: 2×SSC/0.1% SDS at about 37° C. or 42° C. (hybridizationconditions); 0.5×SSC/0.1% SDS at about room temperature (low stringencywash); 0.5×SSC/0.1% SDS at about 42° C. (moderate stringency wash). Anexample of moderately-high stringency hybridization conditions is asfollows: 0.1×SSC/0.1% SDS at about 52° C. (moderately-high stringencywash). An example of high stringency hybridization conditions is asfollows: 0.1×SSC/0.1% SDS at about 65° C. (high stringency wash).

The polynucleotides of the present invention can, if desired: be nakedor be in a carrier suitable for passing through a cell membrane (e.g.,polynucleotide-liposome complex or a colloidal dispersion system),contained in a vector (e.g., retrovirus vector, adenoviral vectors, andthe like), linked to inert beads or other heterologous domains (e.g.,antibodies, ligands, biotin, streptavidin, lectins, and the like), orother appropriate compositions disclosed herein or known in the art.Thus, viral and non-viral means of polynucleotide delivery can beachieved and are contemplated. The polynucleotides of the presentinvention can also contain additional nucleic acid sequences linkedthereto that encode a polypeptide having a distinct functionality, suchas the various heterologous domains set forth herein.

The polynucleotides of the present invention can also be modified, forexample, to be resistant to nucleases to enhance their stability in apharmaceutical formulation. The described polynucleotides are useful forencoding chimeric polypeptides of the present invention, especially whensuch polynucleotides are incorporated into expression systems disclosedherein or known in the art. Accordingly, polynucleotides including anexpression vector are also included.

For propagation or expression in cells, polynucleotides described hereincan be inserted into a vector. The term “vector” refers to a plasmid,virus, or other vehicle known in the art that can be manipulated byinsertion or incorporation of a nucleic acid. Such vectors can be usedfor genetic manipulation (i.e., “cloning vectors”) or can be used totranscribe or translate the inserted polynucleotide (i.e., “expressionvectors”). A vector generally contains at least an origin of replicationfor propagation in a cell and a promoter. Control elements, includingpromoters present within an expression vector, are included tofacilitate proper transcription and translation (e.g., splicing signalfor introns, maintenance of the correct reading frame of the gene topermit in-frame translation of mRNA and stop codons). In vivo or invitro expression of the polynucleotides described herein can beconferred by a promoter operably linked to the nucleic acid. “Promoter”refers to a minimal nucleic acid sequence sufficient to directtranscription of the nucleic acid to which the promoter is operablylinked (see, e.g., Bitter et al., Methods in Enzymology, 153:5 16-544(1987)). Promoters can constitutively direct transcription, can betissue-specific, or can render inducible or repressible transcription;such elements are generally located in the 5′ or 3′ regions of the geneso regulated.

In the present invention, for viruses that bind a co-receptor, it isadvantageous to introduce and express a polynucleotide encoding achimeric polypeptide into the cells that are susceptible to viralinfection (e.g., cells that express the co-receptor). In this way, theexpressed chimeric polypeptide will be secreted by the transformedsusceptible cell in close proximity to the co-receptor, therebyinhibiting or preventing access of the virus to the co-receptor which,in turn, inhibits or prevents viral infection of cells. To this end, atissue-specific promoter can be operably linked to the polynucleotidesequence to confer expression of the chimeric polypeptide in anappropriate target cell.

As used herein, the phrase “tissue-specific promoter” means a promoterthat is active in particular cells or tissues that confers expression ofthe operably linked polynucleotide in the particular cells, e.g., livercells, hematopoietic cells, or cells of a specific tissue within ananimal. The term also covers so-called “leaky” promoters, which regulateexpression of a selected DNA primarily in one tissue, but causeexpression in one or more other tissues as well. An inducible promotercan also be used to modulate expression in cells. “Inducible promoter”means a promoter whose activity level increases in response to treatmentwith an external signal or agent (e.g., metallothionein IIA promoter,heat shock promoter). A “repressible promoter” or “conditional promoter”means a promoter whose activity level decreases in response to arepressor or an equivalent compound. When the repressor is no longerpresent, transcription is activated or derepressed. Such promoters maybe used in combination and also may include additional DNA sequencesthat are necessary for transcription and expression, such as introns andenhancer sequences.

As used herein, the term “operably linked” means that a selectedpolynucleotide (e.g., encoding a chimeric polypeptide) and regulatorysequence(s) are connected in such a way as to permit transcription whenthe appropriate molecules (e.g., transcriptional activator proteins) arebound to the regulatory sequence(s). Typically, a promoter is located atthe 5′ end of the polynucleotide and may be in close proximity of thetranscription initiation site to allow the promoter to regulateexpression of the polynucleotide. However, indirect operable linkage isalso possible when a promoter on a first vector controls expression of aprotein that, in turn, regulates a promoter controlling expression ofthe polynucleotide on a second vector.

When cloning in bacterial systems, constitutive promoters, such as T7and the like, as well as inducible promoters, such as pL ofbacteriophage gamma, plac, ptrp, ptac, may be used. When cloning inmammalian cell systems, constitutive promoters, such as SV40, RSV andthe like, or inducible promoters derived from the genome of mammaliancells (e.g., the metallothionein promoter) or from mammalian viruses(e.g., the mouse mammary tumor virus long terminal repeat, theadenovirus late promoter), may be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences of the invention.

Mammalian expression systems that utilize recombinant viruses or viralelements to direct expression may be engineered. For example, when usingadenovirus expression vectors, the nucleic acid sequence may be ligatedto an adenovirus transcription/translation control complex, e.g., thelate promoter and tripartite leader sequence. Alternatively, thevaccinia virus 7.5K promoter may be used (see, e.g., Mackett et al.,Proc. Natl. Acad. Sci. USA, 79:7415-7419 (1982); Mackett et al., J.Virol., 49:857-864 (1984); Panicali et al., Proc. Natl. Acad. Sci. USA,79:4927-4931 (1982)).

Mammalian expression systems further include vectors specificallydesigned for “gene therapy” methods, including adenoviral vectors (U.S.Pat. Nos. 5,700,470 and 5,731,172), adeno-associated vectors (U.S. Pat.No. 5,604,090), herpes simplex virus vectors (U.S. Pat. No. 5,501,979),and retroviral vectors (U.S. Pat. Nos. 5,624,820, 5,693,508 and5,674,703 and WIPO publications WO92/05266 and WO92/14829). The chimericpolypeptide encoding gene can be introduced into vaccine deliveryvehicles, such as attenuated vaccinia (M. Girard et al., C R Acad SciIII., 322:959-66 (1999); B. Moss et al., AIDS, 2 Suppl 1:S103-5 (1988)),Semiliki-forest virus (M. Girard et al., C R Acad Sci III., 322:959-66(1999); S. P. Mossman et al., J Virol., 70: 19.53-60 (1996)), orSalmonella (R. Powell et al., In: Molecular Approaches to the control ofinfectious diseases, pp. 183-1 87, F. Bran, E. Norrby, D. Burton, and J.Meckalanos (eds), Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1996); M. T. Shata et al., Mol Med Today, 6:66-71 (2000)) to provide anefficient and reliable means for the expression of properly associatedand folded virus coat protein and receptor sequences, for example, gp120and CD4.

Vectors based on bovine papilloma virus (BPV) have the ability toreplicate as extra-chromosomal elements (Sarver et al., Mol. Cell.Biol., 1:486 (1981)). Shortly after entry of an extra-chromosomal vectorinto mouse cells, the vector replicates to about 100 to 200 copies percell. Because transcription of the inserted cDNA does not requireintegration of the plasmid into the host's chromosome, a high level ofexpression occurs. Such vectors also have been employed in gene therapy(U.S. Pat. No. 5,719,054). CMV-based vectors also are included (U.S.Pat. No. 5,561,063).

For yeast expression, a number of vectors containing constitutive orinducible promoters may be used (see, e.g., Current Protocols inMolecular Biology, Vol. 2, Ch. 13, ed. Ausubel et al., Greene Publish.Assoc. & Wiley Interscience (1988); Grant et al., “Expression andSecretion Vectors for Yeast,” in Methods in Enzymology, Vol. 153, pp.516-544, eds. Wu & Grossman, 3 1987, Acad. Press, N.Y. (1987); Glover,DNA Cloning, Vol. II Ch. 3, IRL Press, Wash., D.C. (1986); Bitter,“Heterologous Gene Expression in Yeast,” Methods in Enzymology, Vol.152, pp. 673-684, eds. Berger & Kimmel, Acad. Press, N.Y. (1987); andThe Molecular Biology of the Yeast Saccharomyces, eds. Strathem et al.,Cold Spring Harbor Press, Vols. I and II (1982)). A constitutive yeastpromoter, such as ADH or LEU2, or an inducible promoter, such as GAL,may be used (“Cloning in Yeast,” R. Rothstein, In: DNA Cloning, APractical Approach, Vol. 11, Ch. 3, ed. D. M. Glover, IRL Press, Wash.,D.C. (1986)). Alternatively, vectors that facilitate integration offoreign nucleic acid sequences into a yeast chromosome, via homologousrecombination, for example, are known in the art and can be used. Yeastartificial chromosomes (YAC) are typically used when the insertedpolynucleotides are too large for more conventional yeast expressionvectors (e.g., greater than about 12 kb). The polynucleotides may beinserted into an expression vector for expression in vitro (e.g., usingin vitro transcription/translation kits, which are availablecommercially), or may be inserted into an expression vector thatcontains a promoter sequence that facilitates expression in eitherprokaryotes or eukaryotes by transfer of an appropriate nucleic acidinto a suitable cell, organ, tissue, or organism in vivo.

As used herein, a “transgene” is any piece of a polynucleotide insertedby artifice into a host cell, and becomes part of the organism thatdevelops from that cell. A transgene can include one or more promotersand any other DNA, such as introns, necessary for expression of theselected DNA, all operably linked to the selected DNA, and may includean enhancer sequence. A transgene may include a polynucleotide that ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism. Transgenes may integrate into the host cell's genome or bemaintained as a self-replicating plasmid.

As used herein, a “host cell” is a cell into which a polynucleotide isintroduced that can be propagated, transcribed, or encoded polypeptideexpressed. The term also includes any progeny of the subject host cell.It is understood that all progeny may not be identical to the parentalcell, since there may be mutations that occur during replication. Hostcells include but are not limited to bacteria, yeast, insect, andmammalian cells. For example, bacteria transformed with recombinantbacteriophage polynucleotide, plasmid nucleic acid, or cosmid nucleicacid expression vectors; yeast transformed with recombinant yeastexpression vectors; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV), or transformed with recombinant plasmid expression vectors(e.g., Ti plasmid), insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus), or animal cell systems infectedwith recombinant virus expression vectors (e.g., retroviruses,adenovirus, vaccinia virus), or transformed animal cell systemsengineered for stable expression.

For long-term expression of invention polypeptides, stable expression ispreferred. Thus, using expression vectors containing viral origins ofreplication cells can be transformed with a nucleic acid controlled byappropriate control elements (e.g.,promoter/enhancer sequences,transcription terminators, polyadenylation sites, etc.). Although notwishing to be bound or so limited by any particular theory, stablemaintenance of expression vectors in mammalian cells is believed tooccur by integration of the vector into a chromosome of the host cell.Optionally, the expression vector also can contain a nucleic acidencoding a selectable marker conferring resistance to a selectivepressure or reporter indicating the cells into which the gene has beenintroduced, thereby allowing cells having the vector to be identified,grown, and expanded. As used herein, “reporter gene” means a gene whoseexpression may be assayed; such genes include, without limitation, lacZ,amino acid biosynthetic genes, e.g. the yeast LEI2 gene, luciferase, orthe mammalian chloramphenicol transacetylase (CAT) gene. Reporter genesmay be integrated into the chromosome or may be carried on autonomouslyreplicating plasmids (e.g., yeast 2 micron plasmids). Alternatively, theselectable marker can be on a second vector cotransfected into a hostcell with a first vector containing an invention polynucleotide.

A number of selection systems may be used, including, but not limited tothe neomycin gene, which confers resistance to the aminoglycoside G418(Colberre-Garapin et al., J Mol. Biol., 150: 1 (1981)) and thehygromycin gene, which confers resistance to hygromycin (Santerre et al,Gene, 30: 147 (1984)). Recently, additional selectable genes have beendescribed, namely trpB, which allows cells to utilize indole in place oftryptophan; hisD, which allows cells to utilize histinol in place ofhistidine (Hartman et al., Proc. Natl. Acad. Sci. USA, 85:8047 (1988));and ODC (ornithine decarboxylase), which confers resistance to theornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO(McConlogue, In: Current Communications in Molecular Biology, ColdSpring Harbor Laboratory, ed. (1987)).

As used herein, the term “transformation” means a genetic change in acell following incorporation of a polynucleotide (e.g., a transgene)exogenous to the cell. Thus, a “transformed cell” is a cell into which,or a progeny of which, a polynucleotide has been introduced by means ofrecombinant techniques. Transformed cells do not include an entire humanbeing. Transformation of a host cell may be carried out by conventionaltechniques known to those skilled in the art. When the host cell is aeukaryote, methods of DNA transformation include, for example, calciumphosphate, microinjection, electroporation, liposomes, and viralvectors. Eukaryotic cells also can be co-transformed with inventionpolynucleotide sequences or fragments thereof, and a second DNA moleculeencoding a selectable marker, as described herein or otherwise known inthe art. Another method is to use a eukaryotic viral vector, such assimian virus 40 (SV40) or bovine papilloma virus, to transiently infector transform eukaryotic cells, and express the protein (see, e.g.,Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed.(1982)). When the host is prokaryotic (e.g., E. coli), competent cellsthat are capable of DNA uptake can be prepared from cells harvestedafter exponential growth phase and subsequently treated by the CaCl₂method using procedures well-known in the art. Transformation ofprokaryotes also can be performed by protoplast fusion of the host cell.

Chimeric polypeptides, polynucleotides, and expression vectorscontaining same of the present invention can be encapsulated withinliposomes using standard techniques and introduced into cells or wholeorganisms. Cationic liposomes are preferred for delivery ofpolynucleotides. The use of liposomes for introducing variouscompositions in vitro or in vivo, including proteins andpolynucleotides, is known to those of skill in the art (see, forexample, U.S. Pat. Nos. 4,844,904, 5,000,959, 4,863,740 and 4,975,282).

Liposomes can be targeted to a cell type or tissue of interest by theaddition to the liposome preparation of a ligand, such as a polypeptide,for which a corresponding cellular receptor has been identified. Forexample, in the case of a virus that infects a CD4+ cell, CD4+ cells arean appropriate target and HIV gp120 could be an appropriate ligand forintracellular introduction of a liposome containing a chimericpolypeptide or polynucleotide sequence as described herein. Monoclonalantibodies can also be used for targeting; many such antibodies specificfor a wide variety of cell surface proteins are known to those skilledin the art and are available. The selected ligand is covalentlyconjugated to a lipid anchor in either preformed liposomes or areincorporated during liposome preparation (see Lee & Low, J Biol. Chem.,269:3 198 (1994); Lee & Low Biochem. Biophys. Actu, 1233: 134 (1995)).

The chimeric polypeptides and polynucleotides encoding same of thepresent invention can be introduced into a whole organism. Inparticular, for chimeric polypeptides that contain a virus coatpolypeptide that binds to co-receptor, transgenic animals expressinginvention chimeric polypeptides would be useful for studying thelong-term effects of chimeric expression, as well as determining whetherthe expressed chimeric polypeptide could protect or inhibit infection bya corresponding virus.

Thus, in another embodiment, the invention provides non-human transgenicanimals that express chimeric polypeptides. Preferred animals aresusceptible to viral infection for which a corresponding receptorpolypeptide sequence is known. Preferred animals are those susceptibleto immunodeficiency virus infection, including mammals, such asnon-human primates (e.g., macaques, chimpanzees, apes, gibbons,orangutans, etc.), domestic animals, and livestock, as described herein.

The term “transgenic animal” refers to any animal whose somatic or germline cells bear genetic information received, directly or indirectly, bydeliberate genetic manipulation at the subcellular level, such as bymicroinjection or infection with recombinant virus. The term“transgenic” further includes cells or tissues (i.e., “transgenic cell,”“transgenic tissue”) obtained from a transgenic animal geneticallymanipulated, as described herein. In the present context, a “transgenicanimal” does not encompass animals produced by classical crossbreedingor in vitro fertilization, but rather denotes animals in which one ormore cells receive a recombinant DNA molecule. Transgenic animals can beeither heterozygous or homozygous with respect to the transgene. Methodsfor producing transgenic animals are well known in the art (see, forexample, U.S. Pat. Nos. 5,721,367, 5,695,977, 5650,298, and 5614,396).

The chimeric polypeptides described herein can be used to generateadditional reagents, such as antibodies. Invention antibodies are usefulin the various treatment methods set forth herein. For example, theantibody produced in an immunized subject can protect the subjectagainst virus infection or, alternatively, be transferred to a recipientsubject, thereby passively protecting the second subject againstinfection. Antibodies that bind to an epitope exposed upon complexformation between a virus coat polypeptide sequence and a receptorpolypeptide sequence also can be generated. In addition, inventionantibodies are useful in diagnostic methods, purification methods, andin screening methods (e.g., identifying cryptic epitopes, co-receptors,etc.), as disclosed herein.

Thus, in accordance with the present invention, antibodies that bind tochimeric polypeptides, including antibodies specific for crypticepitopes exposed upon complex formation as set forth herein, areprovided. In one embodiment, the antibody neutralizes multiple viralisolates and viruses from different geographic clades (termed “broadlyneutralizing”) in vitro. In another embodiment, the antibody inhibits,prevents, or blocks virus infection in vitro or in vivo. In variousaspects of these embodiments, the virus neutralized is animmunodeficiency virus, including the HIV-1 and HIV-2 immunodeficiencyviruses set forth herein. Antibody comprising polyclonal antibodies,pooled monoclonal antibodies with different epotopic specificities, anddistinct monoclonal antibody preparations, also are provided.

Antibodies to chimeric polypeptide are produced by administering achimeric polypeptide to an animal. The antibodies can be produced,isolated, and purified using methods well-known in the art. Thus, inanother embodiment, the invention provides methods for producing anantibody to a chimeric polypeptide. A method of the invention includesadministering a chimeric polypeptide to a subject and isolating theantibodies that bind to the chimeric polypeptide. In one embodiment, theantibody produced binds to a cryptic epitope exposed upon the bindingbetween a virus coat polypeptide sequence and a receptor polypeptidesequence.

Preferably, antibodies bind to cryptic epitopes exposed when the viruscoat polypeptide sequence (e.g., envelope polypeptide sequence) and thereceptor polypeptide sequence bind to each other. For example, the HIVenvelope polypeptide sequence gp120 exposes a cryptic epitope uponbinding to CD4 receptor polypeptide sequence, and antibodies to theexposed epitope can lead to broad neutralization of HIV. Such epitopesmay be shared among different viral isolates and geographic cladesaccounting for broad-spectrum neutralizing activity of the antibodiesdirected to these epitopes.

Although not wishing to be bound by theory, it appears that in theabsence of CD4 binding, the cryptic epitope is not exposed or is notantigenic. As used herein, the term “epitope” refers to an antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules, such as amino acids or carbohydrate side chains,and usually have specific three-dimensional structural characteristics,as well as specific charge characteristics. As used herein, the term“cryptic” refers to a property or feature that requires a structural orconformational change for the feature or property to become apparent; inthe absence of the change, the feature or property is “hidden.” Crypticepitopes may be present on either virus coat proteins or receptorpolypeptide sequences.

The term “antibody” includes intact molecules, as well as fragmentsthereof, such as Fab, F(ab′)₂, and Fv, which are capable of binding toan epitopic determinant present in a chimeric polypeptide describedherein. Other antibody fragments are included, so long as the fragmentretains the ability to selectively bind with its antigen. Antibodyfragments (e.g., Fab, F(ab′)₂, and Fv) of the present invention can beprepared by proteolytic hydrolysis of the antibody, for example, bypepsin digestion of whole antibodies. Antibodies which bind to disclosedchimeric polypeptides can be prepared using intact chimeric polypeptideor fragments thereof as the immunizing antigen. In the case of chimericpolypeptide fragments, it is preferred that the virus coat polypeptidesequence and the receptor polypeptide sequence maintain the ability tobind each other so that any cryptic epitopes present will be exposed.The chimeric polypeptide used to immunize an animal is derived fromtranslated polynucleotide or is chemically synthesized and, if desired,can be conjugated to a carrier. Such commonly used carriers chemicallycoupled to the immunizing peptide include, for example, keyhole limpethemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanustoxoid.

Monoclonal antibodies are made by methods well-known to those skilled inthe art (Kohler et al., Nature, 256:495 (1975); and Harlow et al.,Antibodies: A Laboratory Manual, p. 726, eds. Cold Spring Harbor Pub.(1988), which are incorporated herein by reference). Briefly, monoclonalantibodies can be obtained by injecting mice with a compositioncomprising an antigen, verifying the presence of antibody production byanalyzing a serum sample, removing the spleen to obtain B lymphocytes,fusing the B lymphocytes with myeloma cells to produce hybridomas,cloning the hybridomas, selecting positive clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures. Monoclonal antibodies can be isolated and purifiedfrom hybridoma cultures by a variety of well-established techniques,which include, for example, affinity chromatography with Protein-ASepharose, size-exclusion chromatography, and ion-exchangechromatography (see, e.g., Coligan et al., “Production of PolyclonalAntisera in Rabbits, Rats, Mice and Hamsters,” In: Current Protocols inImmunology, §§ 2.7.1-2.7.12 and §§ 2.9.1-2.9.3; and Barnes et al.,“Purification of Immunoglobulin G (IgG),” In: Methods in MolecularBiology, Vol. 10, pp. 79-104, Humana Press (1992)). The preparation ofpolyclonal antibodies is well-known to those skilled in the art (see,e.g., Green et al., “Production of Polyclonal Antisera,” In:Immunochemical Protocols, pp. 1-5, Manson, ed., Humana Press (1992);Harlow et al. (1988), supra; and Coligan et al. (1992), supra §2.4.1,which are incorporated herein by reference).

For therapeutic purposes, antibodies to a chimeric polypeptide producedin one species can be humanized so that the antibody does not induce animmune response when administered to the host, for example, for passiveimmunization. Generally, humanized antibodies are produced by replacinga non-human constant region with a human constant region. Such antibodyhumanization methods are known in the art and are particularly useful inthe methods of the invention (Morrsion et al., Proc. Natl. Acad. Sci.USA, 81:685 1 (1984); Takeda et al., Nature, 314:452 (1985); Singer etal., J. Immunol., 150:2844 (1993)).

Antibodies that bind a chimeric polypeptide, particularly, antibodiesthat bind a cryptic epitope, can neutralize the virus in vitro or invivo (i.e., in a subject). Such antibodies can therefore prevent orinhibit virus infection in vitro or in vivo, and may ameliorate some orall of the symptoms associated with the infection. Such antibodies canbe produced in one subject and then introduced into another, i.e., forpassive immunotherapy. Alternatively, antibodies that bind chimericpolypeptides, when produced in a subject, can protect that subject frominfection or ameliorate some or all of the symptoms associated with theinfection.

Thus, in accordance with the present invention, there are providedmethods for inhibiting, preventing, and ameliorating a viral infectionin a subject. In one embodiment, a method of the invention includesadministering an effective amount of an antibody that binds to achimeric polypeptide to a subject, thereby preventing or inhibitingvirus infection in the subject. In another embodiment, a method of theinvention includes administering an effective amount of a chimericpolypeptide to a subject, thereby producing an immune responsesufficient for preventing or inhibiting virus infection in the subject.In yet another embodiment, a method of the invention includesadministering to a subject an effective amount of a polynucleotideencoding an invention chimeric polypeptide. In various aspects, thechimeric polypeptide contains an immunodeficiency virus envelopepolypeptide, as disclosed herein.

In the methods for inhibiting, preventing, and ameliorating a viralinfection in a subject in which a chimeric polypeptide or apolynucleotide encoding a chimeric polypeptide are administered, animmune response also can be produced. The immune response will likely behumoral in nature, although a administering a polynucleotide encoding achimeric polypeptide may induce a CTL response. It is also understoodthat the methods of the invention can also be used in combination withother viral therapies, as appropriate.

The “effective amount” will be sufficient to inhibit, prevent, orameliorate a viral infection in a subject, or will be sufficient toproduce an immune response in a subject. Thus, an effective amount ofchimeric polypeptide can be that which elicits an immune response to thepolypeptide or a virus upon which the coat protein is based. Aneffective amount administered to a subject already infected with thevirus can also be that which decreases viral load, or increases thenumber of CD4 + cells. An effective amount can be that which inhibitstransmission of the virus from an infected subject to another(uninfected or infected).

In the methods of the invention in which a polynucleotide sequenceencoding a chimeric polypeptide is administered to a subject, a CTLresponse to the chimeric polypeptide can be produced against a virusthat contains the corresponding coat polypeptide sequence.

As the chimeric polypeptides, polynucleotides, and antibodies of thepresent invention will be administered to subjects, including humans,the present invention also provides pharmaceutical formulationscomprising the disclosed chimeric polypeptides, polynucleotides, andantibodies. The compositions administered to a subject will therefore bein a “pharmaceutically acceptable” or “physiologically acceptable”formulation.

As used herein, the terms “pharmaceutically acceptable” and“physiologically acceptable” refer to carriers, diluents, excipients,and the like that can be administered to a subject, preferably withoutexcessive adverse side effects (e.g., nausea, headaches, etc.). Suchpreparations for administration include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions, orsuspensions, including saline and buffered media. Vehicles includesodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present, such as, for example, antimicrobial,anti-oxidants, chelating agents, and inert gases and the like. Variouspharmaceutical formulations appropriate for administration to a subjectknown in the art are applicable in the methods of the invention (e.g.,Remington's Pharmaceutical Sciences, 18^(th) ed., Mack Publishing Co.,Easton, Pa. (1990); and The Merck Index, 12^(th) ed., Merck PublishingGroup, Whitehouse, N.J. (1996)).

Controlling the duration of action or controlled delivery of anadministered composition can be achieved by incorporating thecomposition into particles or a polymeric substance, such as polyesters,polyamine acids, hydrogel, polyvinyl pyrrolidone, ethylene-vinylacetate,methylcellulose, carboxymethylcellulose, protamine sulfate orlactide/glycolide copolymers, polylactide/glycolide copolymers, orethylenevinylacetate copolymers. The rate of release of the compositionmay be controlled by altering the concentration or composition of suchmacromolecules. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, beads, and lipid-based systems,including oil-in-water emulsions, micelles, mixed micelles, andliposomes.

The compositions administered by a method of the present invention canbe administered parenterally by injection, by gradual perfusion overtime, or by bolus administration (for example, in the case of passiveprotection against HIV infection resulting from a needlestick injury) orby a microfabricated implantable device. The composition can beadministered via inhalation, intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity (e.g., vaginal or anal),transdermally, topically, or intravascularly. The compositions can beadministered in multiple doses. The doses or “effective amount” neededfor treating, inhibiting, or preventing viral infection or transmission,or for inducing an immune response, preferably will be sufficient toameliorate some or all of the symptoms of the infection, althoughpreventing progression or worsening of the infection also is asatisfactory outcome for many viral infections, including HIV. Aneffective amount can readily be determined by those skilled in the art(see, for example, Ansel et al., Pharmaceutical Drug Delivery Systems,5^(th) ed. (Lea and Febiger (1990), Gennaro ed.)).

The chimeric polypeptides, polynucleotides, and antibodies of theinvention are also useful for diagnostic purposes. For example, achimeric polypeptide having a virus coat polypeptide sequence derivedfrom a virus that utilizes co-receptor for infection can be used toidentify subjects that express co-receptors having decreased bindingaffinity for the chimeric polypeptide. Subjects which have a decreasedbinding affinity will likely have a decreased risk of infection by thevirus. Alternatively, subjects expressing co-receptors having anincreased binding affinity for the chimeric polypeptide will likely beat increased risk of virus infection. In this way, subjects havingdecreased or increased risk to virus infection can be identified. Forexample, subjects expressing a CCR5 or CXCR4 co-receptor havingincreased or decreased affinity for a chimeric polypeptide comprised ofHIV gp120-CD4 will be at increased or decreased risk of HIV infection,respectively. Accordingly, such methods also are useful for assessingprognosis; subjects expressing a high affinity binding co-receptorlikely having a poorer prognosis.

In the case of the chimeric polypeptides disclosed herein that have avirus coat polypeptide sequence of a virus that utilizes a co-receptor,such chimeric polypeptides are useful for identifying agents thatmodulate binding of the virus to the co-receptor. Such chimericpolypeptides also are useful for identifying agents that modulate theintramolecular interaction/binding of the virus coat polypeptidesequence to the receptor sequence within the chimeric polypeptide. Thus,described chimeric polypeptides that contain coat polypeptide of virusthat may not utilize co-receptor can be used to identify agents thatmodulate binding of the coat sequence to the receptor sequence withinthe chimeric molecule.

Thus, in accordance with the present invention, there are providedmethods for identifying an agent that modulates binding between a virusand a virus co-receptor, and methods for identifying an agent thatmodulates binding between a virus and a virus receptor.

In one embodiment, a method of the invention includes contacting achimeric polypeptide with a co-receptor polypeptide under conditionsallowing the chimeric polypeptide and the co-receptor polypeptide tobind, in the presence and absence of a test agent, and detecting bindingin the presence and absence of the test agent. In another embodiment, amethod of the invention includes contacting a chimeric polypeptide thatforms an intramolecular complex with a test agent, and detecting bindingbetween the virus coat polypeptide sequence and the receptor polypeptidesequence within the chimera. A decreased amount of binding in thepresence of the test agent thereby identifies an agent that inhibitsinteraction/binding between the virus and the virus co-receptor orreceptor. Increased binding in the presence of the test agent therebyidentifies an agent that stimulates interaction/binding between thevirus and the virus co-receptor or receptor.

The contacting can occur in solution, solid phase, on intact cells, orin an organism, such as a non-human primate. In various embodiments, thevirus is an immunodeficiency virus, such as HIV and the co-receptor is achemokine, such as CCR5 or CXCR4. The binding of viruses that utilizeco-receptors for cell penetration is a critical step for subsequentinfection, viral proliferation, and the ultimate pathological symptomsresulting therefrom. Thus, in another embodiment, methods foridentifying agents that inhibit virus cell penetration, infection, andproliferation, as well as agents that ameliorate the symptoms associatedwith the virus infection, are provided. In a method of the presentinvention for identifying such agents, the test agent can be added aftercontacting the chimeric polypeptide with the co-receptor polypeptide or,alternatively, before contacting the chimeric polypeptide with theco-receptor polypeptide.

Candidate agents include antibodies, antivirals, a co-receptorpolypeptide sequence (e.g., from CCR5 or CXCR4), peptidomimeties oractive fragments thereof. Candidate agents also encompass numerouschemical classes, including organic molecules, like small organiccompounds having a molecular weight of more than 50 and less than about2,500 daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl, or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures, and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules, including, but not limited to, peptides,saccharides, fatty acids steroids, purines, pyrimidines, derivatives,structural analogs, or combinations thereof.

Candidate agents are obtained from a wide variety of sources, includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant, and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical, and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc., to producestructural analogs. Where the method detects binding, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin, etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

A variety of other reagents may be included in the assay. These includereagents, like salts, neutral proteins, e.g. albumin, detergents, etc.,that are used to facilitate optimal protein-protein binding and/orreduce non-specific or background interactions. Reagents that improvethe efficiency of the assay, such as protease inhibitors, nucleaseinhibitors, anti-microbial agents, etc., may be used. The mixture ofcomponents are added in any order that provides for the requisitebinding. Incubations are performed at any suitable temperature,typically between 4° C. and 40° C. Incubation periods are selected foroptimum activity, but may also be optimized to facilitate rapidhigh-throughput screening. Typically, between 0.1 and 1 hour will besufficient.

In various embodiments, the virus is an immunodeficiency virus, asdescribed herein, such as HIV, HTLV, SIV, FeLV, FPV, or herpes virus. Inadditional embodiments, the co-receptor is a CCR5, CXCR4, CCR-2b, CCR3,CCR8, V28/CX3CR1, US28 (herpes virus encoded chemokine like receptor),STRL33/BOB/TYMSTR, GPR1 5/Bonzo, or GPR1 polypeptide sequence.

An agent identified by a method of the invention described herein can befurther tested for its ability to inhibit virus binding or infection ofa cell in vitro or in vivo. Thus, in accordance with the presentinvention, there are provided methods for identifying an agent thatinhibits virus infection of a cell. A method of the invention includescontacting a cell susceptible to virus infection with an infectiousvirus particle in the presence and absence of a test agent, anddetermining whether the test agent inhibits virus binding or infectionof the cell, thereby identifying an agent that inhibits virus infection.In various embodiments, the test agent is added before or aftercontacting the cell with the infectious virus particle. The method alsocan be performed in any suitable animal, such as a non-human primate.

The chimeric polypeptides described herein are also useful foridentifying novel co-receptors or characterizing proteins asco-receptors. In this way, viral infection and subsequent pathogenesisfor any virus can be better understood, thereby enabling improvedtreatment of the infection. For example, one method for identifying anovel co-receptor or characterizing co-receptor function is thetwo-hybrid system, which can detect protein-protein interactions throughthe activation of a reporter whose expression is induced by interactingpolypeptides. Thus, an appropriate chimeric polypeptide can be used as abait sequence in a yeast or mammalian two-hybrid system to screen alibrary for the purpose of identifying interacting proteins, includingnovel co-receptors. Well established biochemical methods of detectingprotein-protein interactions (e.g., column chromatography, gradientcentrifugation, co-immunoprecipitation analysis, etc.) also areapplicable in identifying co-receptors or in characterizing proteins ashaving potential co-receptor function.

The chimeric polypeptides that bind co-receptors also are useful foridentifying a co-receptor binding site. For example, by producingco-receptor polypeptide fragments and contacting the fragments with anappropriate chimeric polypeptide. The contacting can be done insolution, (e.g., co-precipitation), solid phase (e.g., affinity column),or on an intact cell (e.g., contacting co-receptor fragments on a cellsurface and detecting whether the co-receptor fragment inhibits chimericpolypeptide binding to the cell). A co-receptor binding site, onceidentified, can be used as an antiviral agent to treat infection, forexample.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and are notintended to be limiting. Other features and advantages of the inventionwill be apparent from the following detailed description, and from theclaims. The invention is further described in the following examples,which do not limit the scope of the invention(s) described in theclaims.

EXAMPLE I

This Example describes the construction of polynucleotides encoding asingle chain gp120-CD4 chimeric polypeptide FLSC, TsSC, FLSC-R/T andRLSC-R/T CD4M9. The strategy for building a single chain complex isbased on the placement of a 20 to 30 amino acid linker sequence betweenthe C terminus of gp120 and the N terminus of CD4. Analyses of thecrystal structure of modified gp120 bound to soluble CD4 and 17b Fab(Dwong, P. D. et al., Nature, 393:648-59 (1998)) using Swiss PDB Viewersuggested that a chimeric molecule should be capable of intramolecularinteractions leading to formation of a gp120-CD4 complex. A single chainnucleic acid encoding a gp120-CD4 chimeric polypeptide (SEQ ID NO: 1)was constructed by arranging the respective coding sequences in thefollowing order: (1) at the 5′ end, a synthetic, codon encoding gp120 ofthe macrophage-tropic HIVs, BaL; (2) a sequence encoding a 20 amino acidlinker consisting of glycines, alanine, and serines; (3) sequences forsoluble CD4 domains 1 and 2 (D1D2); and (4) at the 3′ end, sequencesencoding a short polypeptide derived from the c-myc oncogene for FLSC.The FLSC-R/T nucleotide sequence (SEQ ID NO: 3) encodes for a proteinhaving a mutation at the c-terminal end of gp120 wherein the arginine isreplaced with a threonine (SEQ ID NO: 4). FLSC-R/T CD4M9 (SEQ ID NO: 5)includes further changes in the nucleotide sequence of a chimerapolypeptide (SEQ ID NO: 6) of the present invention wherein the CD4 D1D2region is replaced with a sequence coding for CD4M9 that encodes for apeptide that mimics the functional activity of the CD4 D1D2 region. Thecodon optimized gp120 sequence was used as it permits high-levelexpression in a rev-independent manner (Haas, J., et al., Curr. Biol.,6:3 15-24 (1996)). The human CD4 sequence used was derived from T4-pMV7(Maddon, P. J., et al., Cell, 47:333-48 (1986); NIH AIDS ReagentRepository, Bethesda, Md.). The myc polypeptide sequence allowsconvenient analyses, purification, and other manipulation of thechimeric polypeptide.

Complete polynucleotides comprising these different sequences weregenerated by PCR and inserted into pEF6 (Invitrogen) using the strongelongation factor promoter (EF 1) to drive expression. Restrictionenzyme sites were introduced into this construct (designated pEF6-SCBa1)to permit convenient exchange with other envelope genes of otherimmunodeficiency viruses.

Briefly, FLSC molecule was constructed via PCR using the plasmidspMR1W1-9 and T4-pMV7 as templates. The gp120 forward primer wasGGG-GGT-ACC-ATG-CCC-ATG-GGG-TCT-CTG-CAA-CCG-CTG-GCC (SEQ ID NO:7) andthe reverse primer wasGGG-TCC-GGA-GCC-CGA-GCC-ACC-GCC-ACC-AGA-GGA-TCC-ACG-CTT-CTC-GCG-CTG-CAC-CAC-GCG-GCG-CTT(SEQ ID NO:8). The CD4 forward primer wasGGG-TCC-GGA-GGA-GGT-GGG-TCG-GGT-GGC-GGC-GCG-GCC-GCT-AAG-AAA-GTG-GTG-CTG-GGC-AAA-AAA-GGG-GAT(SEQ ID NO:9) and the reverse primer wasGGG-GTT-TAA-ACT-TAT-TAC-AGA-TCC-TCT-TCT-GAG-ATG-AGT-TTT-GTT-CAG-CTA-GCA-CCA-CGA-TGT-CTA-TTT-TGA-ACT-C(SEQ ID NO: 10). The PCR product was subcloned into pEF6 (Invitrogen,Carlsbad, Calif.) using Kpn1 and Pme1 restriction sites.

To construct the pEF6-TcSC plasmid, the full-length gp120 expressingsequence in pEF6-FLSC was exchanged for a truncated version of the gp120sequence (DC1DC5DV1V2). The truncated gp120 was generated usingGGG-GGT-ACC-ATG-CCC-ATG-GGG-TCT-CTG-CAA-CCG-CTG-GCC-ACC-TTG-TAC-CTG-CTG-GGG-ATG-CTG-GTC-GCT-TCC-TGC-CTC-GGA-AAG-AAC-GTG-ACC-GAG-AAC-TTC-AAC-ATG-TGG(SEQ ID NO: 15) as a forward primer andGGG-GGA-TCC-GAT-CTT-CAC-CAC-CTT-GAT-CTT-GTA-CAG-CTC (SEQ ID NO: 16) as areverse primer. The V1 and V2 regions were deleted usingCTG-TGC-GTG-ACC-CTG-GGC-GCG-GCC-GAG-ATG-AAG-AAC-TGC-AGC-TTC-AAC-ATC-GGC-GCG-GGC-CGC-CTG-ATC-AGC-TGC(SEQ ID NO:17) as a forward primer andGCA-GCT-GAT-CAG-GCG-GCC-CGC-GCC-GAT-GTT-GAA-GCT-GCA-GTT-CTT-CAT-CTC-GCC-CGC-GCC-CAG-GGT-CAC-GCA-CAG(SEQ ID NO: 18) as a reverse primer.

The CD4M9 sequence (SEQ ID NO: 19) used to clone into FLSC R/T CD4M9 wasgenerated by using the 5′ to 3′ primersGCG-GCC-GCT-TGC-AAC-CTG-GCC-CGC-TGC-CAG-CTG-CGC-TGC-AAG-AGC-CTG-GGC-CTG-CTG-GGC-AAG-TGC-GCC-GGC-AGC-TTC-TGC-GCC-TGC-GGC-CCC-TAA-GAA-TTC(SEQ ID NO: 21) as a forward primer andGAA-TTC-TTA-GGG-GCC-GCA-GGC-GCA-GAA-GCT-GCC-GGC-GCA-CTT-GCC-CAG-CAG-GCC-CAG-GCT-CTT-GCA-GCG-CAG-CTG-GCA-GCG-GGC-CAG-GTT-GCA-AGC-GGC-CGC(SEQ ID NO: 22) as a reverse primer and annealing together. Fragmentswere cut with Not1 & BamH1, then subcloned into pEF6-FLSC R/T that hadbeen prepared by cutting with Not1 & BamH1 and gel purified to removethe relieved hD1D2 from the FLSC R/T sequence. Clones were confirmed bysequencing.

The recombinant constructs are shown in FIG. 1. The chimeric recombinantwhich contained the BaL gp120 (SEQ ID NO: 24) sequence with a spacerregion (SEQ ID NO: 11) and CD4D1D2 region (SEQ ID NO: 26) was designatedfull-length single chain (FLSC). A second construct was designed toproduce complexes more closely resembling the molecules used to solvethe gp120 crystal structure. This construct was designated truncatedsingle chain (TcSC) and constructed as with FLSC except that a sequenceencoding ΔC1ΔC5ΔV1V2 gp120 was used in place of the full length codingsequence (SEQ ID NO: 28). Also shown are constructs designated FLSC-R/Twherein the BaL gp120 is mutated at amino acid 506 (SEQ ID NO: 30) andFLSC-R/T CD4M9 comprising sequences SEQ ID NO: 30 and 20. The amino acidsequence of the spacer region shown in this example isGSSGGGGSGSGGGGSGGGAAA (SEQ ID NO: 11)

EXAMPLE II

This Example describes the transfection of cells with the polynucleotideencoding the gp120-CD4 chimeric polypeptide and the characterization ofthe expressed soluble polypeptide. Recombinant pEF6-FLSC or pEF6-TcSCwas transfected into 293 cells using Fugene, according to themanufacturer's protocol (Boehringer-Manheim). Stable transfectants wereobtained by selection with 5 ug/ml blasticidin. A stable cell line(293-SC) was cultured under different conditions, and the production ofchimeric polypeptide evaluated by immunoblot analysis using a mixture ofanti-gp120 monoclonal antibodies (Y. H. Abacioglu et al., AIDS Res. Hum.Retroviruses, 10:371-81 (1994)) or anti- human CD4 polyclonal sera(T4-4) (K. C. Deen et al., Nature, 331:824 (1998); R. L. Willey et al.,J. Viral., 66:226-34 (1992); NIH AIDS Reagent Repository).

Briefly, cell culture supernatants containing the chimeric polypeptidewere collected and boiled in SDS-PAGE loading buffer (75 mM Tris, 2%SDS, 10% glycerol, 0.001% bromphenol blue, pH 8.3). The samples werethen electrophoresed in a 4-20% SDS-polyacrylamide gradient gel. Thegel-fractionated proteins were then transferred to a nitrocellulosemembrane. Non-specific binding sites on the membrane were then blockedfor 30 minutes with 2% non-fat dry milk in tris-buffered saline, pH 7.The membrane was then probed with either anti-CD4 polyclonal rabbit sera(T4-4; NIH AIDS Reagent Repository, Bethesda, Md.) or a mixture ofmurine monoclonal antibody against HIV gp120. As shown in FIG. 2, thetransfected cells expressed a soluble protein of the expected size (150kD). This polypeptide was reactive with both anti-gp120 and anti-CD4antibodies and, thus, represented intact chimeric polypeptide.

In other studies, reactivity with anti-myc antibody was detected furtherconfirming the identity of the 150 kD species as the chimericpolypeptide. In addition to this polypeptide, bands matching theexpected sizes for gp120 and CD4 D1D2/myc tag were observed indicatingthat a portion of the chimeric polypeptide had been cleaved at thespacer. Addition of a biologically compatible protease inhibitor(Pefabloc; Boerhinger-Mannhiem) yielded essentially uncleaved chimericpolypeptide molecules. This suggests that cleavage of gp120-CD4 occursby a serine protease. The amount of gp120-CD4 chimeric polypeptideproduced by the 293-SC cell line was determined using an anti-gp120capture ELISA with sheep anti-gp120 antibody D7324 (InternationalEnzymes), a sheep polyclonal IgG against a highly conserved epitope inthe gp120 C5 region (J. P. Moore, AIDS, 4:297-305 (1990); J. P. Moore etul., J. Virol., 67:863-75 (1992); J. P. Moore et al., AIDS, 4:307-15(1990)), and a gp120 standard curve.

Briefly, 2 ug/ml of D7324 in phosphate-buffered saline was absorbed ontoa plastic plate. Non-specific binding sites were blocked with 2% non-fatdry milk in buffered saline. Saturating concentrations of cell culturesupernatant from the 293-SC line were then added to the plate. Capturedchimeric polypeptides were detected using inactivated human sera fromHIV-infected patients and anti-human IgG conjugated to horse-radishperoxidase. The 293-SC cell line is estimated to secrete approximately 3ug/ml of gp120-CD4 chimeric polypeptide. The 293-SC cell line has beenadapted to grow in serum-free conditions. Because the immunoblottingstudies indicated that there was some cleavage of the gp120-CD4 chimericpolypeptide a sample of purified single chain was crosslinked and thecrosslinked sample analyzed to determine if the gp120 and CD4 moleculesremained associated. Briefly, single chain gp120-CD4 from supernatantsproduced by 293-SC cell line was purified using an immunoaffinitycolumn. The column was constructed by linking anti-gp120 humanmonoclonal antibody A32 to CNBr-activated sepharose 4B(Amersham-Pharmacia Biotech, Piscataway, N.J.). A32 is specific for ahighly discontinuous epitope on gp120, and preferentially recognizesenvelope bound to CD4. Bound gp120-CD4 was eluted with 0.1 M acetic acidpH 2.5, lyophilized, and dialyzed against PBS. Protein concentration wasdetermined by a BCA assay (Bio-Rad, Hercules, Calif.) using themanufacturer's protocol. A 20 ul aliquot of purified gp 120-CD4 was thencrosslinked with 1 mM solution of the homo-bifunctional crosslinker,BS3, and electrophoresed along with uncrosslinked gp 120-CD4 on a 4-20%polyacrylamide gel. The fractionated proteins were transferred tonitrocellulose, immunoblotted with a mixture of anti-gp120 monoclonalantibodies followed by an alkaline-phosphatase labeled anti-mouse IgG,and visualized with a commercial mixture of BCIP/NBT (KPL).

FIG. 3 shows the results of these studies; uncrosslinked gp120-CD4 is inlane 1, and the crosslinked gp120-CD4 is in lane 2. Lane 1 shows thatthe immunoaffinity column purifies both cleaved and uncleavedsingle-chain gp120-CD4. Crosslinking, as shown in lane 2, generates twobroad bands at 150 kDa and 300 kDa, a pattern suggesting that the singlechain gp120-CD4 in solution exists as an associated 150 kDa molecule.The gp120 and CD4 subunits remain associated, even after the cleavageevent. The 300 kDa band indicates that a portion of gp120-CD4 is dimericin solution and may represent single chain molecules that associatethrough intermolecular interactions between the envelope and CD4 domainson separate molecules. The apparent cleavage of the single-chainmolecules into gp120 and CD4 moieties under certain conditions (FIG. 2)might be a concern for DNA vaccines, since such processing couldpotentially occur in vivo. However, these studies show that despitecleavage the single-chain molecules remained associated as gp120-CD4complexes (FIG. 3). To examine the structural properties of the nativeFLSC in greater detail, different concentrations (1 uM-0.03 uM) of thesame protein preparation examined above were covalently crosslinked inPBS in order to fix any multimeric structures existing in solution.Crosslinked material was then analyzed by immunoblot assay with anti-CD4antibody. As shown in FIG. 4, a major protein band (inset; band A) of172 kD was consistently visible along with two minor bands of highermolecular weight. One of the minor bands (inset; band B) had an apparentsize of approximately 302 kD, while the other (inset; band C) failed tomigrate far enough into the gel to allow an accurate assessment of sizeby SDS-PAGE. The appearance and proportions of the different proteinbands were not dependent on the FLSC concentration prior tocrosslinking. Thus, densitometric analyses indicated that bands A, B andC consistently represented approximately 65%, 25% and 10% of the totalprotein, respectively.

In comparison to the FLSC, the chromatographic profile of thecrosslinked TcSC was more complex. Under non-denaturing conditions TcSCeluted as a broad series of peaks ranging from 166 kD to 353 kD. Such aprofile indicated that the shorter TcSC polypeptide forms multiplehigher order structures upon expression and/or purification. Thisbehavior indicates that the TcSC exists primarily as variably sizedchains of polypeptides joined by interactions between gp 120 sequencesand CD4 sequences in separate molecules. Since the TcSC was created bydeleting 20 C-terminal amino acids from gp120, the distance between theCD4 core structure and the CD4bd of gp120 was shortened which may hinderthe ability of the TcSC to achieve an intramolecular gp120-CD4interaction thereby favoring formation of interchain complexes.Nevertheless, TcSC also exhibited the antigenic and functional featuresof a gp120-CD4 complex. It is possible that because of intermolecularinteractions involving multiple TcSC molecules, a smaller proportion ofthe total protein expressed a co-receptor binding site capable ofinteracting with surface co-receptors. Alternatively, deletion of theVI/V2 regions in the TcSC may decrease the relative affinity of the BaLenvelope for CCR5. Further modification of the TcSC to elongate thelinker between the gp120 and CD4 moieties might allow formation of ahigher proportion of intrachain complexes. Whether the multimeric natureof the TcSC puts this molecule at a disadvantage to FLSC remains an openquestion, since studies with other multimeric molecules suggest they aremore potent immunogens than their monomeric counterparts (A. L. DeVicoet al., AIDS Rev., I:4-14 (1999); S. A. Jeffs et al., J. Gen Virol,77:1403-1410 (1996); R. A. LaCasse et al., Science, 283:357-62 (1999)).

EXAMPLE III

This Example describes data demonstrating the binding of gp120-CD4chimeric polypeptide to several different antibodies reactive with gp120and CD4. The binding of gp120 to CD4 causes conformational changes inthe molecule leading to the exposure of the co-receptor-binding domain.Therefore, antibodies directed against epitopes in this domain shouldreact strongly with properly folded single-chain molecules. In order todetermine exposed epitopes in chimeric molecules, antigenic propertiesof FLSC and TcSC molecules were compared. Purified FLSC and TcSC weresubjected to immunochemical analyses by antigen capture ELISA. In brief,BaLgp120, gp120-rsCD4 complexes or single chain chimeric molecules werecaptured using a purified polyclonal sheep antibody (InternationalEnzymes, Fallbrook, Calif.) raised against a peptide derived from theC-terminal 15 amino acids of gp120, D7324 (J. P. Moore et al., AIDS Res.Hum. Retroviruses, 4:369-79 (198X)), adsorbed to the matrix. The D7324was diluted in PBS to 2 ug/ml and adsorbed to 96-well plates (Maxisorbplates, VWR Scientific, St. Louis, Mo.) by incubating overnight at roomtemperature. Plates were treated BLOTTO (5% non-fat dried milk intris-buffered saline) in order to prevent nonspecific binding to thewells. After washing the plates with TBS samples were diluted in BLOTTOand 200 ul aliquots incubated in duplicate D7324-coated wells for 1 hourat room temperature. Bound antigen was detected using a pool ofinactivated HIV-I+ sera diluted 1:1000 in BLOTTO followed by goatanti-human IgG labeled with horseradish peroxidase (KPL, Gaithersburg,Md.).

Detection was also accomplished using monoclonal antibodies (MAbs A32,17b and 48d) previously shown to preferentially bind gp120 afterengagement of CD4 (M. Thali et al., J. Virol., 67:3978-86 (1993)),followed by the appropriate-labeled second antibody. Two of theantibodies, 17b and 48d, bind within the co-receptor attachment sitethat is induced by CD4 binding (N. Sullivan et al., J. Virol.,72:4694-703 (1998); A. Trkola et al, Nature, 384: 184-6 (1996); L. Wu etal., Nature, 384: 179-1 83 (1996)). Antibody C 11, which recognizes aconserved epitope in the C1-C5 region of free gp120, was also tested.Antibodies were diluted in BLOTTO and incubated for 1 hour at roomtemperature. Plate were washed three times with TBS between eachincubation step. The amounts of gp120 sequences present in samples weredetermined based on a standard curve generated with commercialrecombinant HIV IIIB gp120 (Bartels, Issaquah, Wash.). In comparativestudies involving BaLgp120-rsCD4 complexes, D7324-coated plates weretreated with saturating concentrations of gp120. After washing thewells, an excess concentration of rsCD4 (1 ug/ml) was then added to thewells and incubated for 1 hour to form the complexes. In order toevaluate the TcSC antigen which lacks the D7324 epitope, an alternateELISA format using anti-CD4 MAb 45 (Bartels, Issaquah, Wash.) forcapture was developed. The antibody was adsorbed to plastic at 1 ug/mland wells blocked with BLOTTO. Assays were then carried out as aboveusing the indicated human sera or human monoclonal antibodies.

As shown in FIG. 5A, all of the antibodies reacted strongly with theFLSC. However, the half-maximal binding concentrations of antibodies17b, 48d, and A32 were consistently higher with FLSC versus gp120 alone,and equivalent to what was observed with soluble, non-covalentBaLgp120-rsCD4 complexes. The higher immunoreactivity of FLSC wasspecific to the antibodies directed against the CD4-induced epitopes, asthere was no significant difference in the half-maximal bindingconcentrations of antibody C11 with FLSC versus free gp120.

As shown in FIG. 5B, the level of 17b and 48d reactivity with TcSC wasequivalent to what was observed with FLSC analyzed in parallel. Asexpected, antibodies C11 and A32 did not react with TcSC as the bulk oftheir respective epitopes were deleted from the TcSC construct.

The binding of gp120 and CD4 sequences in the single-chain moleculesshould also block exposure of epitopes in the CD4 binding site on gp120.To confirm that such binding had occurred, that the CD4 binding site ofgp120 was no longer available for binding, FLSC and TcSC were evaluatedusing the Mab45 capture format and a series of monoclonal antibodies(IgG1b12, F91, and 205-469) directed against the CD4 binding domain(CD4bd) on gp120.

As shown in FIG. 5C, none of these antibodies reacted with either FLSCor TcSC, although positive reactivity was observed with pooled HIV+ seratested in parallel. This data indicates an interaction between CD4sequences and the gp120 CD4 binding domain present within FLSC and TcSCmolecules.

In sum, these results demonstrate that gp120-CD4 chimeric polypeptidereactivity was comparable to that observed with complexes made bycombining soluble gp120 and CD4 (uncrosslinked), and higher than withgp120 alone. These data indicate that the single-chain gp120-CD4molecules formed interacting complexes similar to the transition stateHIV envelope-CD4 complex. The captured gp120-CD4 was also reactive withanti-CD4 antiserum and anti-myc antibody in other ELISA studies,consistent with the western blot analyses. Taken together, these dataindicate that a majority of the single-chain gp120-CD4 moleculesrepresent properly folded gp120-CD4 complexes.

EXAMPLE IV

This Example describes data demonstrating the binding of gp120-CD4chimeric molecules, containing a CCR5-specific HIV envelope sequence, toCCR5 expressing cells.

The formation of the gp120-CD4 complex normally exposes the envelopedomains that interact with an appropriate co-receptor (M. Thali et al.,J. Virol., 67:3978-86 (1993); M. A. Vodicka et al., Virol., 233: 193-8(1997)). Therefore, another measure of properly folded gp120-CD4complexes and its ability to inhibit virus infection of a cell is theability to bind to a CCR5 co-receptor.

To evaluate the ability of the single-chain complexes to bindco-receptor, purified single-chain gp120-CD4 molecules were allowed tointeract with cells that express either CCR5 or CXCR4. Briefly,supernatants containing gp120-CD4 single-chain were generated bytransient transfection of 293 cells with pEF6-SC. Supernatants were thenadded to an immunoaffinity column of A32 and the purified single-chaineluted with 0.2 M Acetic Acid pH 2.5, and analyzed by D7324-captureELISA and by immunoblot, as described. Fractions containing single chainwere collected, equilibrated to pH 7, and concentrated.

For the binding, the purified single-chain preparation was allowed tointeract with L1.2 cells that express CCR5 (L. Wu et ul., Nature, 384:179-1 83 (1996); L. Wu et al., J. Exp. Med., 186: 1373-8 1 (1997)).L1.2, L1.2/X4, and L 1.2/R5 cells, murine B-cells lines that express noco-receptor, CXCR4, or CCR5 were mixed with decreasing concentrations ofpurified single-chain protein. After incubation at 37° C. for 1 hour,the cells were washed. Bound single-chain molecules were detected with 1ug/ml of MAb C11 (J. E. Robinson et al., J. Cell. Biochem. Suppl.,16E:71 (1992); M. Thali et al., J. Virol., 67:3978-86 (1993), an anti-gp120 MAb, followed by an anti-human IgG that was labeled with afluorescent molecule, phycoerythrin. C11 recognizes a conformationaldeterminant formed by the C1-C4 regions. The level of bound fluorescencewas determined by fluorescence activated cell sorting (FACS) analysiswith a FACS Calibur instrument (Becton Dickinson). The mean fluorescenceintensity for each sample was calculated using the Cell Quest 3.1.3program (Becton Dickinson).

As shown in FIG. 6, both single chain gp120-CD4 complexes (FLSC andTcSC) bound to the CCR5-expressing, but not CXCR4-expressing, L1.2cells. Maximal binding was observed with FLSC at concentrations (10ug/ml) equivalent to what was observed with soluble BaL gp120-rsCD4complexes tested as controls. In comparison, approximately 10-foldhigher concentrations of the TcSC were required to approach saturationbinding. Thus, gp120-CD4 chimeric polypeptide presents functionalco-receptor binding site(s) for CCR5, as expected for a moleculecontaining a macrophage tropic gp120.

The absence of binding to CXCR4 in these studies was not entirelyunexpected in view of the apparent specificity of the HIV envelopepolypeptide in the gp120-CD4 chimera for CD4. Thus, by constructingpolypeptide chimeras that bind to CXCR4 or other co-receptors, or bymodifying a virus coat polypeptide, as described herein, to obtain achimeric polypeptide that binds to another co-receptor, other virus coatpolypeptide-receptor polypeptide chimeras can be obtained that bind toother co-receptors.

To demonstrate that single-chain gp120-CD4 is binding to CCR5 throughits co-receptor binding site, competition binding studies with 17b and48d antibodies, which have been shown to interact with the co-receptorbinding site of gp120 and prevent gp120/sCD4 complexes from interactingwith co-receptor expressing cells, were performed. For controls, anothergp120 antibody, C11, and a gp41 antibody F240, was used. All of theseantibodies are derived from HIV-1 infected patients . Each antibody wasused at 10 ug/ml and added together with 3 ug/ml of purifiedsingle-chain molecule to L1.2 cells that express either CCR5 or CXCR4.Bound gp120-CD4 was detected with C11, followed by anti-human IgGlabeled with PE. The amount of gp120-CD4 was determined by FACS andexpressed as a percentage of the total bound in control wells withoutcompeting antibody.

As shown in FIG. 7, 17b and 48d strongly inhibited the binding of bothsingle-chain complexes to the cells. In the presence of theseantibodies, the binding signal on CCR5-expressing cells was the same asthe background binding seen with L 1.2/CXCR4 and L1.2 parental cells.Interestingly, 2G12, a potent neutralizing antibody, also reduced theinteraction of all complex forms with CCR5. In comparison, anti-gp120antibodies recognizing epitopes outside the co-receptor binding domain,C 11, A32, and an anti-gp41 antibody, F240, all failed to reduce thebinding of FLSC or TcSC to the CCRS-expressing L1.2 cells.

These results indicate that the gp120 co-receptor binding site isimportant for binding to co-receptor. These results also indicate thatagents that inhibit binding/interaction between gp 120-CD4 andco-receptor can be identified using such an assay. Such agents may havepotential value as therapeutics.

In sum, the data demonstrate the successful expression of a soluble,chimeric polypeptide which duplicates the transition state conformationof a virus coat-receptor complex. Given this accomplishment, it is nowpossible to employ the chimeric polypeptide or polynucleotides encodingthe polypeptide for immunization of a subject to produce an immuneresponse to virus or virus having similar coat polypeptide epitopes. Theimmune response produced can be an antibody (humoral) or CTL response.In addition, given the fact that the chimeric polypeptide binds to anappropriate co-receptor on the surface of living cells, the polypeptidecan be administered to subjects acutely exposed to an immunodeficiencyvirus in order to passively protect cells expressing the co-receptorfrom virus infection.

EXAMPLE V

This example describes data demonstrating that a gp 120-CD4 chimericmolecule can neutralize infection by HIV strains using the sameco-receptor. The single-chain molecules were further examined for theirability to neutralize R5 and X4 viruses. A total of 10⁴ U373/CD4/MAGIcells (M. A. Vodicka et al., Virology, 233: 193-8 (1997)) expressingeither CCR5 or CXCR4 were allowed to attach overnight to flat-bottomtissue culture wells. Culture medium was then removed and replaced with100 ul of fresh media containing various concentrations of chimericprotein. An additional 100 ul of media containing 50 TCID₅₀ of virus wasthen added to the culture. The entire mixture was then incubated at 37°C. until syncytia were visible, typically within 3-5 days. Culture wellswere then treated with a P-galactosidase chemiluminescent reagent,Galatostar (Tropix, Bedford, Mass.), according to the manufacturer'sprotocol. Virus infection was determined as a function ofchemiluminescence, quantified using a Victor² fluorescence plate reader(EG&G Wallac, Gaithersburg, Md.). Background signal was determined inassays carried out in the absence of virus. Signals obtained for thetest assays were then corrected by subtracting the background value.Percent infection was calculated by dividing the corrected relativelight units for each experimental well by the corrected light units forcontrol wells containing only cells and virus. The 90% inhibitory dose(ID₉₀) values were determined from plots of test protein concentrationversus percent inhibition of infection. All test conditions were carriedout in triplicate.

As shown in FIG. 8, both FLSC and TcSC potently and selectivelyneutralized the R5 HIV-1 BaL isolate, while there was only a slightinhibition (ID₉₀>10 ug/ml) of 2044 isolate. In comparison, uncomplexedBaLgp120 inhibited entry of both HIV-1BaL and X4 (HIV-12044) viruses asexpected due to its direct interactions with CD4. Thus, the datademonstrate that a virus coat polypeptide-receptor chimeric molecule canbind to a cellular co-receptor thereby blocking binding or infection ofthe cells by virus that utilize the co-receptor for binding orinfection.

EXAMPLE VI

This Example describes the construction and expression of a modifiedgp120-CD4 chimeric polypeptide having an immunoglobulin polypeptidesequence, gp120-CD4-IgG1. This exemplary heterologous domain addsfunctionality to the gp120-CD4 chimeric polypeptide, including adhesinand immunopotentiating functions, prolonging stability, increasingcirculating half-life and ability to cross the placental barrier. Thisexample also shows that the gp120-CD4-IgG1 chimera binds to co-receptorexpressed on the surface of intact cells and neutralizes HIV virus.Gp120, a subunit of the envelope protein of HIV-I binds to CD4 andundergoes a conformational change that permits the complex to interactwith a co-receptor, such as CCR5. This interaction permits the infectionof HIV-1 into target CD4+ cells. Antibodies or other agents thatinterfere with the interaction of HIV-1 with the co-receptor can preventinfection.

To identify such agents, single-chain gp120-CD4 was modified by fusionto the constant regions that form the IgG1 heavy chain, hinge CH2 andCH3 (FIG. 9). Gp120-CD4-IgG1 can be used to identify agents that block,inhibit, or disrupt HW-1 interaction with the co-receptor, therebyidentifying agents that inhibit HIV infection. The gp120-CD4-IgG1polypeptide comprising SEQ ID NOs: 24, 11, 26 and 32 could also be usedas a passive immunotherapeutic to prevent HIV infection after an acuteexposure, such as a needlestick injury.

Two hundred ninety-three cells were transiently transfected with theplasmid containing gp120-CD4-IgG1 comprising at least SEQ ID NOs: 23, 25and 31, and the expressed protein was characterized by immunoblotting ofthe culture supernatants. Briefly, collected supernatant samples wereelectrophoresed onto a 4-20% gradient PAGE gel. Fractionated proteinswere transferred to nitrocellulose and detected with a mixture ofanti-gp120 monoclonal antibodies. As shown in FIG. 10, the transientlytransfected cells expressed gp 120-CD4-IgG1 (lane 1). Supernatant fromcells expressing purified gp120 derived from HIV-1 BaL (lane 2) waselectrophoresed for relative size comparison. The gp120-CD4-IgG1polynucleotide encodes a protein having the predicted size for agp120-CD4-IgG1 heavy-chain chimera. Like the original gp120-CD4, aportion of gp120-CD4-IgG1 is cleaved producing a 120 kDa proteinfragment that is most likely gp120 (“Cleaved gp120”). The size of thisfragment suggests that gp120-CD4-IgG1 is being cleaved within thespacer. To assure that the gp120-CD4-IgG1 is folded into a conformationpermissive for binding co-receptor, dilutions of the supernatant wereadded to L 1.2 cells that express either CCR5 or CXCR4 co-receptors.Bound gp120-CD4-IgG1 was detected with anti-human IgG that was labeledwith Europium, a fluorescent reagent. The amount of fluorescence isdirectly related to the amount of bound material.

As shown in FIG. 11, gp120-CD4-IgG1 binds specifically to L 1.2 cellsthat express CCR5. Again, little binding to CXCR4 was detected usingthis assay, which is consistent with the results for gp120-CD4. Thesestudies indicate that heterologous domains conferring additional orenhanced functionality can be added to chimeric molecules withoutaffecting their ability to form a complex that binds to cellco-receptor. To confirm that binding of chimeric gp120-CD4-IgG1 heavychain to CCR5 expressing cells was mediated by co-receptor binding siteof gp120, binding was studied in the presence of blocking antibody 17b.Briefly, for the MAb/FLSC-IgG1 competition studies, sodium butyrateactivated L1.2 cells expressing co-receptor were added to V-bottomplates at 10⁵/well. 10 ug/ml FLSC-IgG1 and 1 ug/ml MAbs were added tothe cells.

Cells and protein were incubated together for 1 hour at 37° C. Cellswere pelleted and washed with TBS three times. Bound material wasdetected with phytoerytherin-labeled anti-human IgG at 5 ug/ml for 1hour at 4° C. The cells were washed three times with TBS then analyzedby fluorescence-activated cell sorting (FACS).

As shown in FIG. 12, 17b, an antibody that recognizes the CCR5-bindingdomain on gp120, blocks FLSC-IgG1 interaction with L1.2R.5 cells whilecontrol antibody, F240, does not. These data demonstrate that theFLSC-IgG1 interacts with the R5 co-receptor via the R5-binding domain ongp120. To confirm that chimeric gp120-CD4-IgG1 heavy chaicould blockvirus entry into cells, neutralization assays were then performed. Inbrief, U373/CD4/MAGI cells that xpress either CCR5 or CXCR4 were allowedto attach to flat-bottom tissue culture trays overnight at 10⁴cells/well. The medium was removed and varying concentrations of MAbsand immunoadhesins were then added to cells in 100 ul of media. Virus(50 TCID₅₀/well of in 100 ul of media) was then added and the mixtureincubated at 37° C. until syncytia were visible, typically 3-5 days.Plates were read using a P-galactosidase chemiluminescent reagent,Galatostar, according to the manufacturer's protocol and thechemiluminescence produced was quantified using a Victor² as previouslydescribed. Percent virus growth was calculated by using the relativelight units for (experimental well)—background wells with novirus)/(wells with virus but no protein)—(background wells) (Table 2).ID₅₀ and ID₉₀ were determined graphically. TABLE 2 Neutralization of X4,R5, and X4/R5 HIV by FLSC-IgGl U373/CD4/CCR5 FLSC-IgGl 2G12 2F5 1lgGlbl2 Control 1gG ID90 (ug/mL) BaL 3.1 >10 >10 1.57 >10 ADA4.58 >10 >10 >10 >10 89.6 3.56 8.07 >10 3.39 >10 U373/CD4/CXCR4 SClg2G12 2F5 lgGlbl2 Control 1gG ID90 (ug/mL) 2044 >10 >10 >10 1.57 >102005 >10 >10 >10 >10 >10 89.6 >10 >10 >10 5.34 >10The data in Table 2 indicate that FLSC-IgG blocks viruses that use R5for cell entry.

FLSC-IgG neutralizes virus as effective as 2G12, 2F5, and IgG1b12,antibodies that are currently being evaluated in passive immunotherapytrials. These data therefore further affirm the usefulness of gp120-CD4chimeras to inhibit HIV infection in particular, and the applicabilityof virus coat protein-receptor chimeras as inhibitors of other virusesthat utilize co-receptor for binding or cell penetration in general.

EXAMPLE VII

This Example describes data demonstrating that mutation of the furincleavage site improves the stability of the FLSC complex. The positionof the cleavage site that separates the FLSC fragments is probablylocated within the C terminal gp 120 sequences present only in FLSC,since the shorter TcSC did not exhibit degradation. Notably, thesesequences encompass the gp120 gp41 junction normally cleaved by thefurin protease (M. Girard et al., C R Acad Sci III., 322:959-66)(1999)). Cleavage of the FLSC at the natural furin site would beconsistent with the behavior of the FLSC fragments, as it would haveminimal impact on the structures of the gp120 and CD4 moieties and theircapacity to interact.

In order to determine if this putative furin site accounts for cleavage,BaLgp120, BaLgp120 complexed with an sCD4 molecule consisting of thefirst two domains (V1V2) of CD4, FLSC, and FLSC R/T were captured ontoplastic via an antibody specific for the C-terminus of gp120 (antibodybinding was unaffected by the R/T mutation). Four domain V1-V4 sCD4 weretitrated onto the captured complexes starting at 30 ug/ml. Four domainsCD4 has a higher affinity for gp120 than the two domain V1V2 and,therefore, would compete off the smaller unit from complexes. Bound fourdomain CD4 was detected with antibody OKT4, which only binds the fourdomain CD4. The results in FIG. 13 show that mutation of the furincleavage site prevents the V1 V2 found on the FLSC R/T from dissociatingas readily as the cleaved FLSC, thus improving its stability of the FLSCR/T complex. Introduction of the R?T mutation into the BaLgp 120c-terminus eliminates the furin mediated cleavage observed with theFLSC. Reducing this cleavage improves the continuity of the linkersequence and improves the stability of the FLSC construct (see FIG. 13)by increasing the local concentration of the gp120 and CD4 moieties. Theexperimental result of this increase is the reduction in the ability ofthe soluble four domain CD4 to compete with the two domain CD4 found onthe FLSC R/T.

EXAMPLE VIII

This Example describes the transfection of cells with the polynucleotideencoding the gp120-CD4 modified chimeric polypeptide and thecharacterization of the expressed soluble polypeptide. RecombinantpEF6-FLSC, pEF6-RLSC-R/T, pEF6-FLSC-R/T CD4M9 and pEF6-BaLgp120 weretransfected into 293 cells using Fugene, according to the manufacturer'sprotocol (Boehringer-Manheim). Stable transfectants were obtained byselection with 5 ug/ml blasticidin. Briefly, cell culture supernatantscontaining the chimeric polypeptides were collected and boiled inSDS-PAGE loading buffer (75 mM Tris, 2% SDS, 10% glycerol, 0.001%bromphenol blue, pH 8.3). The samples were then electrophoresed in a4-20% SDS-polyacrylamide gradient gel. The gel-fractionated proteinswere then transferred to a nitrocellulose membrane. Non-specific bindingsites on the membrane were then blocked for 30 minutes with 2% non-fatdry milk in tris-buffered saline, pH 7. The membrane was then probedwith a mixture of murine monoclonal antibody against HIV gp120 and boundantibodies were detected with alkaline phosphatase labeled goatanti-mouse IgG.

As shown in FIG. 14, the BaLgp120 (Lane 1) and the FLSC-R/T CD4M9 (Lane4) migrated with an approximate molecular weight of 120 kDa. While theFLSC R/T CD4M9 is predicted to be approximately 130 kDa, the differenceof 10 kDa is difficult to see on this blot. The FLSC (lane 2) is a 150kDa protein that is cleaved at the furin site at the c-terminus of theprotein. This cleavage separates the gp120 and CD4 components of theFLSC. The lower 120 kDa band is the result of this cleavage. Thereleased CD4 component is not visible on this blot because theantibodies used to detect the proteins were specific for gp120. Theapparent cleavage of the single-chain molecules into gp120 and CD4moieties under certain conditions might be a concern for DNA vaccines,since such processing could potentially occur in vivo. This Exampledescribes data demonstrating that mutation of the furin cleavage siteimproves the stability of the FLSC complex. The position of the cleavagesite that separates the FLSC fragments is probably located within the Cterminal gp12O sequences present only in FLSC. Notably, these sequencesencompass the gp120/gp41 junction normally cleaved by the furin protease(M. Girard et al., C R Acad Sci III., 322:959-66 (1999)). Cleavage ofthe FLSC at the natural furin site would be consistent with the behaviorof the FLSC fragments, as it would have minimal impact on the structuresof the gp120 and CD4 moieties and their capacity to interact. Theresults show that mutation of the furin cleavage site prevents the V1 V2found on the FLSC R/T from dissociating as readily as the cleaved FLSC,thus improving the stability of the FLSC R/T complex. As a result, theR/T mutation used to create FLSC R/T minimizes this cleavage andstabilizes the protein.

EXAMPLE IX

This Example describes data demonstrating the binding of gp120-CD4chimeric polypeptide to an antibodies reactive with gp120 and CD4. Thebinding of gp120 to CD4 causes conformational changes in the moleculeleading to the exposure of the co-receptor-binding domain. Therefore,antibodies directed against epitopes in this domain should reactstrongly with properly folded single-chain molecules. In order todetermine exposed epitopes in chimeric molecules, antigenic propertiesof BaLgp120, FLSC, FLSC-R/T and FLSC-R/T CDM9 molecules were compared.Detection was accomplished using monoclonal antibodies 17b previouslyshown to preferentially bind gp120 after engagement of CD4 (M. Thali etal., J. Virol., 67:3978-86 (1993)), followed by the appropriate-labeledsecond antibody. The antibody 17b, a human monoclonal antibody thatrecognizes an epitope that becomes increasingly exposed when gp120interacts with CD4 and binds within the co-receptor attachment site(CCR5). (N. Sullivan et al., J. Virol., 72:4694-703 (1998); A. Trkola etal, Nature, 384: 184-6 (1996); L. Wu et al., Nature, 384: 179-1 83(1996)). Antibodies were diluted in BLOTTO and incubated for 1 hour atroom temperature. Plate were washed three times with TBS between eachincubation step. The amounts of gp120 sequences present in samples weredetermined based on a standard curve generated with commercialrecombinant HIV gp120 (Bartels, Issaquah, Wash.). The antibody wasadsorbed to plastic at 1 ug/ml and wells blocked with BLOTTO. Assayswere then carried out as above using the indicated human monoclonalantibodies.

As shown in FIG. 16, the binding curves of 17b with BaLgp120, FLSC,FLSC-R/T and FLSC-R/T CDM9 molecules were enhanced by binding of 17b toFLSC-R/T or FLSC chimeric proteins both of which contain both gp120 andCD4. 17b also binds to FLSC-R/T CD4M9 with the efficiency equivalent tothat of FLSC-R/T indicating that the 17b epitope is exposed in theFLSC-R/T CD4M9 protein. Taken together, these data indicate that thesingle chain gp120-CD4 molecules FLSC, FLSC-R/T and FLSC-R/T CDM9represent properly folded gp120-CD4 complexes.

EXAMPLE X

This Example describes data demonstrating the binding of gp120-CD4chimeric molecules, containing a CCR5-specific HIV envelope sequence, toCCR5 expressing cells. The formation of the gp120-CD4 complex normallyexposes the envelope domains that interact with an appropriateco-receptor (M. Thali et al., J. Virol., 67:3978-86 (1993); M. A.Vodicka et al., Virol., 233: 193-8 (1997)). Therefore, another measureof properly folded gp120-CD4 complexes and its ability to inhibit virusinfection of a cell is the ability to bind to a CCR5 co-receptor.

To evaluate the ability of the single-chain complexes to bindco-receptor, purified single-chain gp120-CD4 molecules were allowed tointeract with canine thymocytes, Cf2Th, that either express CCR5 or haveno co-receptor. Briefly, supernatants containing gp120-CD4 single-chainschimeric polypeptides FLSC-R/T and FLSC-R/T CDM9 molecules weregenerated by transient transfection of 293 cells with pEF6.

For the binding, the purified single-chain preparation was allowed tointeract with canine thymocytes that express CCR5 or have noco-receptor. Bound single-chain molecules were detected with anti-gp120MAb, A32, followed by PE-labeled goat anti-human IgG that was labeledwith a fluorescent molecule, phycoerythrin. The level of boundfluorescence was determined by fluorescence activated cell sorting(FACS) analysis with a FACS Calibur Instrument (Becton Dickinson). Theamount of fluorescence is directly related to the amount of boundmaterial. The mean fluorescence intensity for each sample was calculatedusing the Cell Quest 3.1.3 program (Becton Dickinson). The results shownin FIG. 15 show that the FLSC-R/T CD4M9 bind to the CCR5 expressingcells but not to cells without a co-receptor with the efficiencyequivalent to that of FLSC-R/T.

EXAMPLE XI

This example describes neutralization of primary R5 HIV-1 (92BR020) bysera from FLSC-inoculated mice. C587B1/6 mice were inoculated four timeswith 25 ug of FLSC per mouse mixed with 10 ug cholera toxin (CT).Inoculation occurred at two week intervals. 14 days after the lastinoculation, sera from the individual mice were collected and assayedfor neutralizing activity against primary R5 HIV-1 isolate 92BR020.Serial dilutions of sera starting at 1:2 were mixed with 50 TCID₅₀infection doses of virus/well and 10⁴ U373/CD4/R5/MaGI cells/well. After24 hours, the sera, virus and media were replaced with 200 ul of freshmedia. The assay was allowed to incubate for 5 days until syncytia werevisible. Growth of HIV-1 was indicated by production of b-galactosidasein cell lysates as measured using a chemiluminescent reagent,Galactostar (Tropix) according to manufacture's protocol. Virusinfection was determined as a function of chemiluminescence, quantifiedusing a Victor² (EG&G Wallac, Gaithersburg, Md.) fluorescence platereader. Background signal was determined with assays carried out in theabsence of virus and sera. Signal obtained for the test assays were thencorrected by subtracting the background value. The percent invention wascalculated by dividing the corrected relative light units for eachexperimental well by the corrected light units for control wellscontaining only cells and virus. Sera from the FLSC inoculated mice arelabeled #0, #1, #2, #3, #4, and naive mouse is labeled “C”.

As shown in FIG. 17, as the dilution factor is increased there is alsoan increase in virus infection. Additionally, the sera isolated fromcontrol mouse showed no effect on virus infection, while highconcentrations of sera from mouse #2 showed a minimal amount of virusinfection.

In sum, the data demonstrate the successful expression of a soluble,chimeric polypeptide which duplicates the transition state conformationof a virus coat-receptor complex. Given this accomplishment, it is nowpossible to employ the chimeric polypeptide or polynucleotides encodingthe polypeptide for immunization of a subject to produce an immuneresponse to virus or virus having similar coat polypeptide epitopes. Theimmune response produced can be an antibody (humoral) or CTL response.In addition, given the fact that the chimeric polypeptide binds to anappropriate co-receptor on the surface of living cells, the polypeptidecan be administered to subjects acutely exposed to an immunodeficiencyvirus in order to passively protect cells expressing the co-receptorfrom virus infection.

EXAMPLE XII

FLSC and complexes of BaLgp120 and sCD4 were captured onto D7324-coatedELISA plates. D7324 is a sheep polyclonal IgG that is reactive to theC-terminal region of gp120 and is an antibody that is commonly used toexamine the antigenicity of HIV-1 envelope proteins by capture-ELISA.BaLgp120/sCD4 complexes were then crosslinked for 30 mins with 0.5 mMBis(sulfosuccinimidyl)suberate (Pierce), then treated with 10 mMTris-HCL to stop the reaction. BaLgp120/sCD4 & FLSC plates were thenwashed with TBS. Monoclonal antibodies against the V3 loop (39F), C1-C5(C11), C1-C4 (A32), coreceptor binding domain (17b), and C3-V4 (2G12)regions of BaLgp120 were titrated onto the captured antigens. Boundantibodies were detected with goat-anti-human IgG labeled withhorse-radish peroxidase. FIG. 18 shows that the crosslinking reactionalters the structure of the BaLgp120/sCD4 complex and reduces theantigenicity of the 39F, C11, A32 and 17b epitopes. In contrast, theseepitopes are not occluded on the FLSC. This antigenic alteration wouldimpact the function of these epitopes. For instance, the epitoperecognized by 17b interacts with the R5 coreceptor. Occlusion of thisepitope by the crosslinker would reduce the ability of the crosslinkedcomplex to interact with the coreceptor. This observation would alsosuggest that that crosslinked complex could not be used to screen forreagents that may potentially block HIV-1 via its coreceptor.

EXAMPLE XIII

Purified R/T FLSC-IgG1 was crosslinked for 30 mins with 0.5 mMBis(sulfosuccinimidyl)suberate (Pierce), then treated with 10 mMTris-HCL to stop the reaction. Crosslinked material was then compared touncrosslinked material run in reducing and non-reducing SDS-PAGEconditions. As shown in FIG. 19, the uncrosslinked material on thereducing gel (middle lane) runs at 180 kDa, the expected size of theBaLgp120-CD4-IgG1 chimera. The smaller band is the appropriate size ofCD4-IgG indicating that the chimera is cleaved between the BaLgp120 andthe CD4-IgG portion of the molecule. This observation suggests thatalthough the R/T mutation eliminates the cleavage due to furin-protease,another protease can act on the c-terminus of gp120. The uncrosslinkedmaterial in non-reducing conditions (right lane) runs at 360 kDa, thepredicted size of the fully assembled immunoadhesin. This observationindicates that while a portion of the material is cleaved (see middlelane) immunoadhesin remains associated. Crosslinking of the material,which stabilizes the assemble structure, confirms this observation (leftlane). Here the material runs approximately 360 kDa as expected. Ahigher molecular weight form is also visible suggesting that a portionof purified preparation is aggregated.

EXAMPLE XIV

293 cells were transiently transfected either pcDNA-human CCR5 orpcDNA-rhesus CCR5 or no plasmid 24 hrs prior to use. Transfected cells(10⁵/well) were incubated at 37° C. for 1 hr with the indicatedconcentration of R/T FLSC-IgG1. Bound R/T FLSC-IgG1 was detected withphycoerythrin conjugated Goat anti-human IgG and analyzed by FACS. FIG.20 shows that R/T FLSC-IgG1 binds to both human and rhesus CCR5.

Canine thymocytes expressing CCR5 (CF2Th-R5) (10⁵) were incubated with 3ug/mL R/T LSC-IgG1 and the indicated concentration of chemokine for 1 hrat 37° C. Bound R/T FLSC-IgG1 was detected using phycoerythrinconjugated goat anti-human IgG and analyzed by FACS. RANTES is aCCR5-specific chemokine and as expected competes with R/T FLSC-IgG1 forthe receptor. SDF, a CXCR4-specific chemokine, was used a control. FIG.21 provides further proof that the R/T FLSC-IgG1 may be used as ascreening tool to define reagents that may block HIV-1 infection via itscoreceptor, CCR5.

All references cited herein are incorporated by reference herein for allthat they teach and for all purposes. It is to be understood that, whilethe invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1. A polynucleotide sequence comprising a nucleic acid sequence encodinga chimeric polypeptide selected from the group consisting of SEQ ID NO:13, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO:
 6. 2. A polynucleotidesequence comprising a nucleic acid sequence encoding a chimericpolypeptide comprising: a virus coat polypeptide sequence and a viralreceptor polypeptide sequence, wherein the coat polypeptide sequence andthe receptor polypeptide sequence are linked by an amino acid spacer ofsufficient length to allow the coat polypeptide sequence and the viralreceptor polypeptide sequence to bind to each other wherein the viruscoat polypeptide sequence is selected from the group consisting of SEQID NO: 24, SEQ ID NO: 30, and SEQ ID NO:
 28. 3. The polynucleotidesequence according to claim 2, where the receptor polypeptide sequenceis selected from the group consisting of SEQ ID NO: 26 and SEQ ID NO:20.
 4. The polynucleotide sequence according to claim 1, wherein thenucleic acid sequence is selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO:
 12. 5. Thepolynucleotide sequence according to claim 2, wherein a nucleic acidsequence for the virus coat polypeptide is selected from the groupconsisting of SEQ ID NO: 23, SEQ ID NO: 29 and SEQ ID NO:
 27. 6. Thepolynucleotide sequence according to claim 5, wherein a nucleic acidsequence for the receptor polypeptide is selected from the groupconsisting of SEQ ID NO: 25 and SEQ ID NO:
 19. 7. The polynucleotidesequence according to claim 6, further comprising a nucleotide sequenceencoding for a heterologous domain.
 8. The polynucleotide sequenceaccording to claim 7, wherein the heterologous domain is an IgG ordomains thereof.
 9. An expression vector comprising at least onenucleotide sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 23, SEQ ID NO: 29,SEQ ID NO: 27, SEQ ID NO: 25 and SEQ ID NO:
 19. 10. An isolated hostcell containing the expression vector of claim
 7. 11. An antibody orfunctional fragment thereof that binds to the chimeric polypeptide ofclaim
 1. 12. An antibody or functional fragment thereof that binds tothe chimeric polypeptide of claim
 2. 13. The antibody of claim 12,wherein the antibody binds to an epitope produced by binding of thevirus coat polypeptide sequence and the receptor polypeptide sequence.14. The antibody of claim 13, wherein the antibody reduces virusinfection.
 15. The antibody of claim 11, wherein the antibody binds toan epitope produced by binding of the virus coat polypeptide sequenceand the receptor polypeptide sequence.
 16. A method for producing animmune response to a HIV virus in a subject comprising administering tothe subject an effective amount of the chimeric polypeptide of claim 2to produce the immune response to the HIV virus.
 17. The methodaccording to claim 16, wherein the subject is human.
 18. The methodaccording to claim 16, wherein the immune response produced is comprisedof antibodies.
 19. The method according to claim 16, wherein the immuneresponse induced comprises cell-mediated effectors selected fromchemokines, cytotoxic T lymphocytes, T helper cells, or cytokines.
 20. Amethod for reducing HIV infection in a subject comprising administeringto the subject an effective amount of the polynucleotide according toclaim
 2. 21. A method for identifying an agent that inhibits aninteraction between a virus and a virus receptor comprising the stepsof: (a) contacting the chimeric polypeptide of claim 2 with a testagent; and (b) detecting binding between the virus coat polypeptidesequence and the viral receptor polypeptide sequence, wherein adecreased amount of binding in the presence of the test agent identifiesan agent that inhibits binding between the virus and the virus receptor.22. A polynucleotide sequence comprising a nucleic acid sequenceencoding a chimeric polypeptide comprising: a virus coat polypeptidesequence and a viral receptor polypeptide sequence, wherein the viruscoat polypeptide sequence and the viral receptor polypeptide sequenceare linked by an amino acid spacer of sufficient length to allow thevirus coat polypeptide sequence and the viral receptor polypeptidesequence to bind to each other, wherein the virus coat polypeptide isHIV gp120 or fragments or variants thereof and the viral receptorpolypeptide is CD4 or fragments or variants thereof.
 23. A chimericpolypeptide comprising: a virus coat polypeptide sequence and a viralreceptor polypeptide sequence, wherein the virus coat polypeptidesequence and the viral receptor polypeptide sequence are linked by anamino acid spacer of sufficient length to allow the virus coatpolypeptide sequence and the viral receptor polypeptide sequence to bindto each other, wherein the virus coat polypeptide is HIV gp120 orfragments or variants thereof and the viral receptor polypeptide is CD4or fragments or variants thereof.